Patent Grant
11643456
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 28, 2019, is named G3004-00809_SL.txt and is 137,284 bytes in size.
The present disclosure relates to human antibodies and binding fragments thereof to carbohydrate antigens, as well as nucleic acids encoding such antibodies, complementary nucleic acids, polypeptides, vectors, host cells and methods of making and using thereof, including pharmaceutical compositions comprising said antibody and/or binding fragments. Further, methods are provided for administering antibodies to a subject in an amount effective to inhibit cancer cells. Specifically, antibodies that bind to stage-specific embryonic antigen 3 (SSEA-3), stage-specific embryonic antigen 4 (SSEA-4) and Globo H are disclosed herein, as well as related compositions and methods of use. Methods of use include, without limitation, cancer therapies and diagnostics.
Recent advances in the isolation, culture and expansion of human B cells are enabling the isolation of large numbers of human antibodies to be used for cancer diagnostics and therapeutics. For several decades, mouse monoclonal antibodies were isolated using the hybridoma technology. However, the therapeutic application of these antibodies was limited by induction of anti-mouse antibodies and autoreactivity. More recently, monoclonal antibodies have been isolated through phage display libraries produced from humans with a humoral response of interest (Mao S, et al. (1999) Proc Natl Acad Sci USA; 96:6953-6958.). Although this technique has produced numerous useful antibodies, its applicability is limited by differences in binding properties between antibodies expressed in bacterial and eukaryotic cells. In addition, phage display may result in heavy- and light-chain combinations that do not occur in the same B cell in vivo.
Numerous surface carbohydrates are expressed in malignant tumor cells. For example, the carbohydrate antigen Globo H (Fucα1→2 Galβ1→>3 GalNAcβ1→3 Galα1→4 Galβ1→4 Glc) was first isolated as a ceramide-linked Glycolipid and identified in 1984 from breast cancer MCF-7 cells. (Bremer E G, et al. (1984) J Biol Chem 259:14773-14777). Previous studies have also shown that Globo H and stage-specific embryonic antigen 3 (2Gal β1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1) (SSEA-3, also called Gb5) were observed on breast cancer cells and breast cancer stem cells (WW Chang et al. (2008) Proc Natl Acad Sci USA, 105(33): 11667-11672). In addition, SSEA-4 (stage-specific embryonic antigen-4) (Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→>4Glcβ1) has been commonly used as a cell surface marker for pluripotent human embryonic stem cells and has been used to isolate mesenchymal stem cells and enrich neural progenitor cells (Kannagi R et al. (1983) EMBO J, 2:2355-2361). Thus, it is of great interest to identify glycan markers associated with and/or predictive of cancers, and develop human monoclonal antibodies against the markers for use in diagnosing and treating a broad spectrum of cancers.
Accordingly, the present disclosure is based on the discovery that Globo series antigens (Globo H, SSEA-3 and SSEA-4) are aberrantly expressed in a broad spectrum of cancers, but not on normal cells. Thus, human monoclonal antibodies to Globo series antigens (Globo H, SSEA-3 and SSEA-4) can address the unmet need for effective treatment and/or prevention for cancer. Cancer cells expressing Globo series antigens can include, but are not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, oral cancer, esophageal cancer, stomach cancer, liver cancer, bile duct cancer, pancreatic cancer, colon cancer, kidney cancer, cervical cancer, ovarian cancer and prostate cancer.
In one aspect, the present disclosure is directed to antibodies or binding fragments thereof specific to Globo series antigens.
In order to generate anti-Globo series antigens human monoclonal antibodies, human B cells are isolated from peripheral blood of vaccinated subjects, plated at a density of one cell per well and cultured for secreted IgG production. The secreted IgGs are assayed for Globo H, SSEA-3 or SSEA-4 binding specificities. Genes encoding Ig VH, Ig Vκ or Ig Vλ from positive wells are recovered using RT-PCR and cloned into expression vectors for generating anti-Globo H, SSEA-3 or SSEA-4 human monoclonal antibody. In one embodiment, the light chains of the antibody is kappa type. In one embodiment, the light chain of the antibody is lamda type.
In one aspect, the present disclosure provides an antibody, and/or an antigen-binding fragment thereof, comprising: a heavy chain variable domain (VH) comprising respective CDRs as disclosed herein and an amino acid sequence of at least about 80%, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 98, or 99% sequence homology to the amino acid sequences as disclosed herein and/or a light chain variable domain (VL) comprising respective CDRs as disclosed herein and an amino acid sequence of at least about 80%, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 98, or 99% sequence homology to the amino acid sequences as disclosed herein respectively.
In one aspect, the present disclosure provides an antibody, or an antigen-binding fragment thereof capable of targeting the Globo-Series carbohydrate antigen, comprising: three heavy chain CDRs and corresponding three light chain CDRs of each respective clones as disclosed herein such as the ones disclosed in Tables 1-28.
In another aspect, the present disclosure provides the antibody or antigen-binding fragment thereof comprising: the heavy chain variable domain comprising an amino acid sequence having at least about 80% identity to the full length heavy chain sequences of each clone as disclosed herein in Tables 1-28 further comprising the three corresponding heavy chain complementarity determining regions (CDRs), CDR1, CDR2 and CDR3 sequences of the said corresponding clone; and the light chain variable domain comprising an amino acid sequence having at least about 80% identity to the full length light chain sequences of said clone as disclosed herein in Tables 1-28 further comprising the three corresponding light chain complementarity determining regions (CDRs), CDR1, CDR2 and CDR3 sequences of said corresponding clone.
For example, the present disclosure provides an antibody, or an antigen-binding fragment thereof capable of targeting the Globo-Series carbohydrate antigen, comprising: a three heavy chain CDRs of SEQ ID NOs: 257, 258 and 259 or conservatively modified amino acid substitutions; and/or b. three light chain CDRs of SEQ ID NOs: 260, 261, and 262 or conservatively modified amino acid substitutions. In another embodiment, the antibody or antigen-binding fragment thereof of the above, comprising: the light chain variable domain comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 3 further comprising the three heavy chain complementarity determining regions (CDRs), CDR1, CDR2 and CDR3 (SEQ ID Nos 257, 258, 259); and/or the light chain variable domain comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 4 further comprising the three light chain complementarity determining regions (CDRs), CDR1, CDR2 and CDR3 (SEQ ID Nos: 260, 261, 262). The same can be repeated for each of the clones recited in Tables 1-28 with the respective full length heavy chain and light chain sequences of each clone and their respective corresponding heavy chain and light chain CDRs.
In certain embodiments, the antibody or antigen-binding fragment thereof is selected from: (a) a whole immunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab′)2; or (e) a disulfide linked Fv.
In certain embodiments, the antibody is an IgG or IgM.
In one aspect, the present disclosure provides a pharmaceutical composition, comprising:
an antibody or an antigen-binding fragment thereof; and at least one pharmaceutically acceptable carrier.
In certain embodiments, the pharmaceutical composition further comprising at least one additional therapeutic agent.
In one aspect, the present disclosure provides a method for inhibiting the proliferation of cancer cells, comprising the administering of an effective amount of an exemplary pharmaceutical composition to a subject in need thereof, wherein the proliferation of cancer cells is inhibited and/or decreased.
In certain embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the exemplary human antibody described herein.
In certain embodiments, the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophageal cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer.
In one aspect, the present disclosure provides a method for staging cancer in a subject, comprising:
(a) applying one or more antibodies that detect expression of Globo series antigens to a cell or tissue sample obtained from the subject;
(b) assaying the binding of the one or more antibodies to the cell or the tissue sample;
(c) comparing the binding with a normal control to determine the presence of the cancer in the subject; and
(d) categorizing disease progression stage based on relative levels of corresponding antibody binding compared to normal baseline index.
In one aspect, the present disclosure provides a method for inhibiting the proliferation of cancer cells, comprising the administering of an effective amount of an pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof targeting Globo-series carbohydrate antigens to a subject in need thereof, wherein the proliferation of cancer cells is inhibited. In one embodiment, the subject is human.
In one aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject in need thereof an effective amount of the antibody or an antigen-binding fragment thereof targeting Globo-series carbohydrate antigens.
In one embodiment, the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer. In one embodiment, the subject is human.
In one aspect, the present disclosure provides a method for cancer diagnosis in a subject, comprising:
(a) Applying one or more antibodies or binding fragments as disclosed herein that detect expression of a panel of markers to a cell or sample obtained from the subject;
(b) Assaying the binding of the one or more antibodies to the cell or the sample; and
(c) Comparing the binding with a normal control to determine the presence of the cancer in the subject.
In one embodiment, the markers consisting of Globo-H, SSEA-3 or SSEA-4.
In one embodiment, the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer.
In one embodiment, the cell is cancer stem cell.
In another embodiment, the sample consists serum, blood, plasma, cells, cell medium, saliva, urine, lymph node fluid, tumor biopsy or tissue culture. In one embodiment, the subject is human.
In one aspect, the present disclosure provides a method of imaging a subject comprising:
(a) Administering an effective amount of an antibody or an antigen-binding fragment thereof as disclosed herein wherein the antibody or an antigen-binding fragment is conjugated to an imaging agent; and (b) Detecting the imaging agent in the subject.
In one embodiment, the imaging agent is a fluorophore, a dye, an MRI contrast agent or a radionuclide.
In one embodiment, the subject has a cancer, the method further defined as a method of detecting a cancer metastasis. In one embodiment, the subject is human.
In one aspect, the present disclosure provides a method of isolating an antibody, or an antigen-binding fragment in a subject, comprising:
(a) Administering to the subject a therapeutically effective dose of Globo series antigens vaccine and a pharmaceutically acceptable carrier:
(b) Collecting a sample from the subject;
(c) Isolating B cells from the sample; and
(d) Cultivating and screening the B cells which bind to the Globo series antigens.
In one embodiment, the Globo series antigens comprising Globo-H, SSEA-3 or SSEA-4. In one embodiment, the subject is human.
In one embodiment, the sample consists serum, blood, plasma, cells, cell medium, lymph node fluid, tumor biopsy or tissue culture.
In one aspect, the present disclosure provides an antibody-drug conjugate (ADC) comprising a drug conjugated to an antibody or an antigen-binding fragment that binds Globo series antigens, wherein VH selected from SEQ ID No: 3, SEQ ID No: 7, SEQ ID No: 11, SEQ ID No: 15, SEQ ID No: 19, SEQ ID No: 23, SEQ ID No: 27, SEQ ID No: 31, SEQ ID No: 35, SEQ ID No: 39, SEQ ID No: 43, SEQ ID No: 47, SEQ ID No: 51, SEQ ID No: 55, SEQ ID No: 59, SEQ ID No: 63, SEQ ID No: 67, SEQ ID No: 71, SEQ ID No: 75, SEQ ID No: 79, SEQ ID No: 83, SEQ ID No: 87, SEQ ID No: 91, SEQ ID No: 95, SEQ ID No: 99, SEQ ID No: 103, or SEQ ID No: 107 and VL selected from SEQ ID No: 4, SEQ ID No: 8, SEQ ID No: 12, SEQ ID No: 16, SEQ ID No: 20, SEQ ID No: 24, SEQ ID No: 28, SEQ ID No: 32, SEQ ID No: 36, SEQ ID No: 40, SEQ ID No: 44, SEQ ID No: 48, SEQ ID No: 52, SEQ ID No: 56, SEQ ID No: 60, SEQ ID No: 64, SEQ ID No: 68, SEQ ID No: 72, SEQ ID No: 76, SEQ ID No: 80, SEQ ID No: 84, SEQ ID No: 88, SEQ ID No: 92, SEQ ID No: 96, SEQ ID No: 100, SEQ ID No: 104, or SEQ ID No: 108.; and wherein the drug is covalently conjugated to the antibody or the antigen-binding fragment by a linker.
In one embodiment, the Globo series antigens comprising Globo-H, SSEA-3 or SSEA-4.
In one embodiment, the linker comprising a p-nitrophenyl linker, a 4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazide (MMCCH) linker, a maleimidocaproyl (MC) linker or a maleimidomethyl cyclohexane-1-carboxylate (MCC) linker. In one embodiment, the drug is a chemical compound or a biological agent. In one embodiment, the drug is an anti-proliferative agent.
In one embodiment, the anti-proliferative agent is selected from cyclophosphamide, opiate, granulocyte colony-stimulating factor (GCSF), estrogen inhibitors (tamoxifen or Fareston), aromatase inhibitors (Arimidex, Aromasin or Femara), pituitary downregulators (Zoladex or Lupron), Novaldex (tamoxifen selective estrogen-receptor modulator), Evista (rolaxifene), Faslodex (estrogen receptor down-regulator), anticoagulant (Refludan), enzyme (Elitek), Hematopoietic growth factor, anti-neoplastic Agent (antimetabolites, miscellaneous cytotoxic agents, vinca alkaloid, Epipodophyllotoxins, Alkylating agents, Taxanes, Antitumor antibiotics, Camptothecins, Nitrosoureas), HER1/EGFR tyrosine kinase inhibitor (Tarceva), VEGF protein inhibitor (Avastin), HER-2/ErbB2 inhibitor (Tyverb/Tykerb), Interferon, Interleukin, Monoclonal antibody, or Glucocorticoid steroid.
In one embodiment, the anti-proliferative agent is selected from erlotinib (TARCEVA); docetaxel (TAXOTERE); gemcitabine (GEMZAR); cisplatin; carboplatin; paclitaxel (TAXOL); trastuzumab (HERCEPTIN); temozolomide (TEMODAL); tamoxifen (NOLVADEX, ISTUBAL, VALODEX); doxorubicin (ADRIAMYCIN); oxaliplatin (ELOXATIN); bortezomib (VELCADE); sutent (SUNITINIB); letrozole (FEMARA); imatinib mesylate (GLEEVEC); MEK inhibitor (Exelixis); fulvestrant (FASLODEX); leucovorin (folinic acid); rapamycin (RAPAMUNE); lapatinib (TYKERB); lonafarnib (SARASAR); sorafenib (NEXAVAR); gefitinib (IRESSA); irinotecan (CAMPTOSAR); tipifarnib (ZARNESTRA); ABRAXANE (Cremophor-free); paclitaxel; vandetanib (ZACTIMA); chloranmbucil; temsirolimus (TORISEL); pazopanib; canfosfamide (TELCYTA); thiotepa; cyclosphosphamide (CYTOXAN, NEOSAR); 5-fluorouracil (5-FU); vinorelbine (NAVELBINE); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA); ibandronate; topoisomerase inhibitor RFS 2000; -difluoromethylornithine (DMFO); tamoxifen (NOLVADEX); raloxifene; droloxifene, 4-hydroxytamoxifen; trioxifene; keoxifene; onapristone; FARESTON (toremifine citrate); 4(5)-imidazoles; aminoglutethimide; MEGASE (megestrol acetate); AROMASIN (exemestane); formestanie; fadrozole; RIVISOR® (vorozole); FEMARA (letrozole); ARIMIDEX (anastrozole); flutamide; nilutamide; bicalutamide; leuprolide; goserelin; troxacitabine (α-1,3-dioxolane nucleoside cytosine analog); lipid kinase inhibitor; oblimersen (GENASENSE); ANGIOZYME; ALLOVECTIN; LEUVECTIN; VAXID; PROLEUKIN; LURTOTECAN; ABARELIX; bevacizumab (AVASTIN); alemtuzumab (Campath); bevacizumab (AVASTIN); cetuximab (ERBITUX); panitumumab (VECTIBIX); rituximab (RITUXAN); pertuzumab (OMNITARG); trastuzumab (HERCEPTIN); tositumomab (Bexxar, Corixia); gemtuzumab; or ozogamicin (MYLOTARG).
In one aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject in need thereof an effective amount of the ADC as disclosed herein.
In one embodiment, the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer. In one embodiment, the subject is human.
The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appending claims.
A more complete understanding of the invention may be obtained by reference to the accompanying drawings, when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments.
Accordingly, antibody methods and compositions directed to the markers for use in diagnosing and treating a broad spectrum of cancers are provided. Anti-Globo series antigens human antibodies were developed and disclosed herein. Methods of use include, without limitation, cancer therapies and diagnostics. The antibodies described herein can bind to a broad spectrum of Globo series antigens-expressing cancer cells, thereby facilitating cancer diagnosis and treatment. Cells that can be targeted by the antibodies include carcinomas, such as those in skin, blood, lymph node, brain, lung, breast, mouse, esophagus, stomach, liver, bile duct, pancreas, colon, kidney, cervix, ovary, prostate cancer, etc.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlow and Lane s (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).
As used herein, the term “glycan” refers to a polysaccharide, or oligosaccharide. Glycan is also used herein to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans usually consist solely of O-glycosidic linkages between monosaccharides. For example, cellulose is a glycan (or more specifically a glucan) composed of ß-1,4-linked D-glucose, and chitin is a glycan composed of ß-1,4-linked N-acetyl-D-glucosamine. Glycans can be homo or heteropolymers of monosaccharide residues, and can be linear or branched. Glycans can be found attached to proteins as in glycoproteins and proteoglycans. They are generally found on the exterior surface of cells. O- and N-linked glycans are very common in eukaryotes but may also be found, although less commonly, in prokaryotes. N-Linked glycans are found attached to the R-group nitrogen (N) of asparagine in the sequon. The sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except praline.
As used herein, the term “antigen” is defined as any substance capable of eliciting an immune response.
As used herein, the term “immunogenicity” refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.
As used herein, the term “epitope” is defined as the parts of an antigen molecule which contact the antigen binding site of an antibody or a T cell receptor.
As used herein, the term “vaccine” refers to a preparation that contains an antigen, consisting of whole disease-causing organisms (killed or weakened) or components of such organisms, such as proteins, peptides, or polysaccharides, that is used to confer immunity against the disease that the organisms cause. Vaccine preparations can be natural, synthetic or derived by recombinant DNA technology.
As used herein, the term “antigen specific” refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.
As used herein, the term “specifically binding,” refers to the interaction between binding pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be embodied by an affinity constant of about 10−6 moles/liter, about 10−7 moles/liter, or about 10−8 moles/liter, or less.
The phrase “substantially similar,” “substantially the same”, “equivalent”, or “substantially equivalent”, as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values, anti-viral effects, etc.). The difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the value for the reference/comparator molecule.
The phrase “substantially reduced,” or “substantially different”, as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.
“Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.
The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “recombinant vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
“Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotides(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
“Oligonucleotide,” as used herein, generally refers to short, generally single-stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which generally lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
The terms “antibody” and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, monovalent, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein). An antibody can be chimeric, human and/or affinity matured.
The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of heavy or light chain of the antibody. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.
Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably, to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.
“Antibody fragments” comprise only a portion of an intact antibody, wherein the portion retains at least one, and as many as most or all, of the functions normally associated with that portion when present in an intact antibody. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody. For example, such an antibody fragment may comprise an antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. Such monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technologies (See, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO98/24893; WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
Antibodies of the present invention can also include chimerized monoclonal antibodies generated from antibodies of the present invention.
The antibodies can be full-length or can comprise a fragment (or fragments) of the antibody having an antigen-binding portion, including, but not limited to, Fab, F(ab′)2, Fab′, F(ab)′, Fv, single chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragment (e.g., Ward et al, Nature, 341:544-546 (1989)), an CDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single chain antibodies produced by joining antibody fragments using recombinant methods, or a synthetic linker, are also encompassed by the present invention. Bird et al. Science, 1988, 242:423-426. Huston et al, Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883.
The antibodies or antigen-binding portions thereof of the present invention may be monospecific, bi-specific or multispecific.
All antibody isotypes are encompassed by the present invention, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE (all classes and subclasses are encompassed by the present invention). The antibodies or antigen-binding portions thereof may be mammalian (e.g., mouse, human) antibodies or antigen-binding portions thereof. The light chains of the antibody may be of kappa or lambda type.
Thus, anti-cancer antibodies of the present invention include in combination with a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, of non-murine origin, preferably of human origin, which can be incorporated into an antibody of the present invention.
Antibodies with a variable heavy chain region and a variable light chain region that are at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86% o, at least about 87%>, at least about 88%>, at least about 89%>, at least about 90%>, at least about 91>, at least about 92%>, at least about 93%>, at least about 94%>, at least about 95%), at least about 96%>, at least about 97%>, at least about 98%>, at least about 99%> or about 100% homologous to the variable heavy chain region and variable light chain region of the antibody produced by the reference antibody, and can also bind to a carbohydrate antigen (e.g. Globo H, SSEA-3 or SSEA-4). Homology can be present at either the amino acid or nucleotide sequence level.
As used herein, substantially “homology” and/or “homologous sequences” of proteins of the invention include, without limitation, conservative amino acid substitutions, or for example alterations that do not effect the VH, VL or CDR domains of the antibodies, e.g., include scFv antibodies where a different linker sequence is used or antibodies where tag sequences or other components are added that do not contribute to the binding of antigen, or alterations to convert one type or format of antibody molecule or fragment to another type or format of antibody molecule or fragment (e.g., conversion from Fab to scFv or vice versa), or the conversion of an antibody molecule to a particular class or subclass of antibody molecule (e.g., the conversion of an antibody molecule to IgG or a subclass thereof, e.g., IgG1 or IgG3).
A “conservative amino acid substitution”, as used herein, is one in which the amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Homology may be assessed by any convenient method. However, for determining the degree of homology between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson et al., 1994). If desired, the Clustal W algorithm can be used together with BLOSUM 62 scoring matrix (Henikoff and Henikoff, 1992) and a gap opening penalty of 10 and gap extension penalty of 0.1, so that the highest order match is obtained between two sequences wherein at least 50% of the total length of one of the sequences is involved in the alignment. Other methods that may be used to align sequences are the alignment method of Needleman and Wunsch (1970), as revised by Smith and Waterman (1981) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Other methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (1988) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.
Generally, computer programs can be employed for such calculations. Programs that compare and align pairs of sequences, like ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990) and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984) are also useful for this purpose. Furthermore, the Dali server at the European Bioinformatics institute offers structure-based alignments of protein sequences (Holm, 1993; 1995; 1998).
The antibodies or antigen-binding portions may be peptides. Such peptides can include variants, analogs, orthologs, homologs and derivatives of peptides, that exhibit a biological activity, e.g., binding of a carbohydrate antigen. The peptides may contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), peptides with substituted linkages, as well as other modifications known in the art.
Also within the scope of the invention are antibodies or antigen-binding portions thereof in which specific amino acids have been substituted, deleted or added. In an exemplary embodiment, these alternations do not have a substantial effect on the peptide's biological properties such as binding affinity. In another exemplary embodiment, antibodies may have amino acid substitutions in the framework region, such as to improve binding affinity of the antibody to the antigen. In yet another exemplary embodiment, a selected, small number of acceptor framework residues can be replaced by the corresponding donor amino acids. The donor framework can be a mature or germline human antibody framework sequence or a consensus sequence. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science, 247: 1306-1310 (1990). Cunningham et al, Science, 244: 1081-1085 (1989). Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994). T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989). Pearson, Methods Mol. Biol. 243:307-31 (1994). Gonnet et al., Science 256: 1443-45 (1992).
The antibody, or antigen-binding portion thereof, can be derivatized or linked to another functional molecule. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent interaction, etc.) to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, a protein or peptide that can mediate association with another molecule (such as a streptavidin core region or a polyhistidine tag), amino acid linkers, signal sequences, immunogenic carriers, or ligands useful in protein purification, such as glutathione-S-transferase, histidine tag, and staphylococcal protein A. One type of derivatized protein is produced by crosslinking two or more proteins (of the same type or of different types). Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, 111. Useful detectable agents with which a protein can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting, exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, and, phycoerythrin. A protein or antibody can also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase and the like. A protein can also be derivatized with a prosthetic group (e.g., streptavidin/biotin and avidin/biotin).
Nucleic acids encoding a functionally active variant of the present antibody or antigen-binding portion thereof are also encompassed by the present invention. These nucleic acid molecules may hybridize with a nucleic acid encoding any of the present antibody or antigen-binding portion thereof under medium stringency, high stringency, or very high stringency conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which is incorporated herein by reference. Specific hybridization conditions referred to herein are as follows: 1) medium stringency hybridization conditions: 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 2) high stringency hybridization conditions: 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and 3) very high stringency hybridization conditions: 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.
A nucleic acid encoding the present antibody or antigen-binding portion thereof may be introduced into an expression vector that can be expressed in a suitable expression system, followed by isolation or purification of the expressed antibody or antigen-binding portion thereof. Optionally, a nucleic acid encoding the present antibody or antigen-binding portion thereof can be translated in a cell-free translation system. U.S. Pat. No. 4,816,567. Queen et al, Proc Natl Acad Sci USA, 86: 10029-10033 (1989).
The present antibodies or antigen-binding portions thereof can be produced by host cells transformed with DNA encoding light and heavy chains (or portions thereof) of a desired antibody. Antibodies can be isolated and purified from these culture supernatants and/or cells using standard techniques. For example, a host cell may be transformed with DNA encoding the light chain, the heavy chain, or both, of an antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding, e.g., the constant region.
As used herein, “substantially purified” or “substantially isolated” refers to a molecule (e.g. a compound) in a state that it is separated from substantially all other molecules normally associated with it in its native state. Preferably, a substantially purified molecule is the predominant species present in a preparation. Particularly, a substantially purified molecule may be greater than 60% free, preferably 75% free, or 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% free, or any range between any two recited percentages free from the other molecules (exclusive of solvent) present in the natural mixture.
The present nucleic acids can be expressed in various suitable cells, including prokaryotic and eukaryotic cells, e.g., bacterial cells, (e.g., E. coli), yeast cells, plant cells, insect cells, and mammalian cells. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC). Non-limiting examples of the cells include all cell lines of mammalian origin or mammalian-like characteristics, including but not limited to, parental cells, derivatives and/or engineered variants of monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2/0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells. The engineered variants include, e.g., glycan profile modified and/or site-specific integration site derivatives.
The present invention also provides for cells comprising the nucleic acids described herein. The cells may be a hybridoma or transfectant.
Alternatively, the present antibody or antigen-binding portion thereof can be synthesized by solid phase procedures well known in the art. Solid Phase Peptide Synthesis: A Practical Approach by E. Atherton and R. C. Sheppard, published by IRL at Oxford University Press (1989). Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed. M. W. Pennington and B. M. Dunn), chapter 7. Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984). G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 1 and Vol. 2, Academic Press, New York, (1980), pp. 3-254. M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984).
The term “hypervariable region”, “HVR”, or “HV”, when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
“Framework” or “FW” residues are those variable domain residues other than the hypervariable region residues as herein defined.
The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO93/1161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).
A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein.
An “affinity matured” antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).
A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
An “agonist antibody”, as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.
A “disorder” is any condition that would benefit from treatment with an antibody of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer.
The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.
“Tumor” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.
As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. “Treating” or “treating” is referred to herein as administration of a therapeutic composition to a subject with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate a disorder, symptoms of the disorder, a disease state secondary to the disorder, or predisposition toward the disorder. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing or decreasing inflammation and/or tissue/organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or disorder.
An “individual” or a “subject” is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, the vertebrate is a human.
“Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In certain embodiments, the mammal is human.
An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
A “therapeutically effective amount” of a substance/molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.
Antibodies Targeting Globo Series Antigens
One aspect of the present disclosure features the new antibody targeting the Globo series antigens (Globo H, SSEA-3, SSEA-4).
Cancers expressing Globo series antigens (SSEA-4, Globo H or SSEA-3) include, but are not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, oral cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, pancreas cancer, colon cancer, kidney cancer, cervix cancer, ovary cancer and prostate cancer.
“SSEA-4 moiety” is defined herein to be a glycan (i.e., a molecule containing a sugar moiety) that is SSEA-4 or a fragment or analog thereof. SSEA-4 is a glycan containing the hexasaccharide epitope (Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1), and optionally, a non-sugar moiety. Its fragment is a glycan containing a fragment of the hexasaccharide epitope and, if applicable, the non-sugar moiety.
“Globo H moiety” is defined herein to be a glycan (i.e., a molecule containing a sugar moiety) that is Globo H or a fragment or analog thereof. Globo H is a glycan containing the hexasaccharide epitope (Fucα1→2 Galβ1→3 GalNAcβ1→3 Galα1→4 Galβ1→4 Glc), and optionally, a non-sugar moiety. Its fragment is a glycan containing a fragment of the hexasaccharide epitope and, if applicable, the non-sugar moiety.
“SSEA-3 moiety” is defined herein to be a glycan (i.e., a molecule containing a sugar moiety) that is SSEA-3 or a fragment or analog thereof. SSEA-3 is a glycan containing the pentasaccharide epitope (Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1), and optionally, a non-sugar moiety. Its fragment is a glycan containing a fragment of the hexasaccharide epitope and, if applicable, the non-sugar moiety.
Exemplars and their amino acid and nucleic acid structures/sequences are provided below:
One aspect of the present disclosure features the new antibodies specific to SSEA-4. The anti-SSEA-4 antibody binds to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα 1→4Galβ1→4Glcβ1.
One aspect of the present disclosure features the new antibodies specific to SSEA-3. The anti-SSEA-3 antibody binds to 2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1.
One aspect of the present disclosure features the new antibodies specific to Globo H. The anti-Globo H antibody binds to Fucα1→2 Galβ1→3 GalNAcβ1→3 Galα1→4 Galβ1→4 Glc.
Any of the antibodies described herein can be a full length antibody or an antigen-binding fragment thereof. In some examples, the antigen binding fragment is a Fab fragment, a F(ab′)2 fragment, or a single-chain Fv fragment. In some examples, the antigen binding fragment is a Fab fragment, a F(ab′)2 fragment, or a single-chain Fv fragment. In some examples, the antibody is a human antibody, a chimeric antibody, or a single-chain antibody.
Any of the antibodies described herein has one or more characteristics of: (a) is a recombinant antibody, a monoclonal antibody, a chimeric antibody, a human antibody, an antibody fragment, a bispecific antibody, a monospecific antibody, a monovalent antibody, an IgG1 antibody, an IgG2 antibody, or derivative of an antibody; (b) is a human, murine, or chimeric antibody, antigen-binding fragment, or derivative of an antibody; (c) is a single-chain antibody fragment, a multibody, a Fab fragment, and/or an immunoglobulin of the IgG, IgM, IgA, IgE, IgD isotypes and/or subclasses thereof; (d) has one or more of the following characteristics: (i) mediates ADCC and/or CDC of cancer cells; (ii) induces and/or promotes apoptosis of cancer cells; (iii) inhibits proliferation of target cells of cancer cells; (iv) induces and/or promotes phagocytosis of cancer cells; and/or (v) induces and/or promotes the release of cytotoxic agents; (e) specifically binds the tumor-associated carbohydrate antigen, which is a tumor-specific carbohydrate antigen; (f) does not bind an antigen expressed on non-cancer cells, non-tumor cells, benign cancer cells and/or benign tumor cells; and/or (g) specifically binds a tumor-associated carbohydrate antigen expressed on cancer stem cells and on normal cancer cells.
Preferably the binding of the antibodies to their respective antigens is specific. The term “specific” is generally used to refer to the situation in which one member of a binding pair will not show any significant binding to molecules other than its specific binding partner (s) and e.g. has less than about 30%, preferably 20%, 10%, or 1% cross-reactivity with any other molecule other than those specified herein.
Immunization of Host Animals and Hybridoma Technology
In one embodiment, the present invention provides for a method for making a hybridoma that expresses an antibody that specifically binds to a carbohydrate antigen (e.g., Globo H). The method contains the following steps: immunizing an animal with a composition that includes a carbohydrate antigen (e.g., Globo H); isolating splenocytes from the animal; generating hybridomas from the splenocytes; and selecting a hybridoma that produces an antibody that specifically binds to Globo H. Kohler and Milstein, Nature, 256: 495, 1975. Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988.
In one embodiment, carbohydrate antigen is used to immunize mice subcutaneously. One or more boosts may or may not be given. The titers of the antibodies in the plasma can be monitored by, e.g., ELISA (enzyme-linked immunosorbent assay) or flow cytometry. Mice with sufficient titers of anti-carbohydrate antigen antibodies are used for fusions. Mice may or may not be boosted with antigen 3 days before sacrifice and removal of the spleen. The mouse splenocytes are isolated and fused with PEG to a mouse myeloma cell line. The resulting hybridomas are then screened for the production of antigen-specific antibodies. Cells are plated, and then incubated in selective medium. Supernatants from individual wells are then screened by ELISA for human anti-carbohydrate antigen monoclonal antibodies. The antibody secreting hybridomas are replated, screened again, and if still positive for anti-carbohydrate antigen antibodies, can be subcloned by limiting dilution.
Exemplary Polyclonal antibodies against the anti-Globo series antigens antibodies may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method. Polyclonal antibodies include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies may be isolated from the serum.
Polyclonal antibodies are generally raised in host animals (e.g., rabbit, mouse, horse, or goat) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, etc.
Any mammalian animal may be immunized with the antigen for producing the desired antibodies. In general, animals of Rodentia, Lagomorpha, or Primates can be used. Animals of Rodentia include, for example, mouse, rat, and hamster. Animals of Lagomorpha include, for example, rabbit. Animals of Primates include, for example, a monkey of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkey, baboon, and chimpanzees.
Methods for immunizing animals with antigens are known in the art. Intraperitoneal injection or subcutaneous injection of antigens is a standard method for immunization of mammals. More specifically, antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc. If desired, the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion, and then administered to mammalian animals. Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining 1 mg or 1 μg of the peptide or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's incomplete adjuvant.
Animals can be boosted until the titer plateaus by several administrations of antigen mixed with an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days. Animals are boosted with ⅕ to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. An appropriate carrier may also be used for immunization. After immunization as above, serum is examined by a standard method for an increase in the amount of desired antibodies. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
In some embodiments, antibodies can be made by the conventional hybridoma technology. Kohler et al., Nature, 256:495 (1975). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or rabbit, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that can specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro.
To prepare monoclonal antibodies, immune cells are collected from the mammal immunized with the antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion. The immune cells used for cell fusion are preferably obtained from spleen. Other preferred parental cells to be fused with the above immunocyte include, for example, myeloma cells of mammalians, and more preferably myeloma cells having an acquired property for the selection of fused cells by drugs.
Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
The above immunocyte and myeloma cells can be fused according to known methods, for example, the method of Milstein et al. (Galfre et al., Methods Enzymol. 73:3-46, 1981). Lymphocytes are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin, and thymidine containing medium). The cell culture is typically continued in the HAT medium for several days to several weeks, the time being sufficient to allow all the other cells, with the exception of the desired hybridoma (non-fused cells), to die. Then, the standard limiting dilution is performed to screen and clone a hybridoma cell producing the desired antibody.
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay. Measurement of absorbance in enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/or immunofluorescence may be used to measure the antigen binding activity of the antibody of the invention. In ELISA, the antibody of the present invention is immobilized on a plate, protein of the invention is applied to the plate, and then a sample containing a desired antibody, such as culture supernatant of antibody producing cells or purified antibodies, is applied. Then, a secondary antibody that recognizes the primary antibody and is labeled with an enzyme, such as alkaline phosphatase, is applied, and the plate is incubated. Next, after washing, an enzyme substrate, such as p-nitrophenyl phosphate, is added to the plate, and the absorbance is measured to evaluate the antigen binding activity of the sample. A fragment of the protein, such as a C-terminal or N-terminal fragment may be used in this method.
Applying any of the conventional methods, including those described above, hybridoma cells producing antibodies that bind to epitopes described herein can be identified and selected for further characterization.
After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. For example, the obtained hybridomas can be subsequently transplanted into the abdominal cavity of a mouse and the ascites are harvested.
The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the protein of the present invention is coupled. The antibody of the present invention can be used not only for purification and detection of the protein of the present invention, but also as a candidate for agonists and antagonists of the protein of the present invention. In addition, this antibody can be applied to the antibody treatment for diseases related to the protein of the present invention.
Activity Assays
Antibodies of the invention can be characterized for their physical/chemical properties and biological functions by various assays known in the art.
Antibodies, or antigen-binding fragments, variants or derivatives thereof of the present disclosure can also be described or specified in terms of their binding affinity to an antigen. The affinity of an antibody for a carbohydrate antigen can be determined experimentally using any suitable method (see, e.g., Berzofsky et al, “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and methods described herein). The measured affinity of a particular antibody-carbohydrate antigen interaction can vary if measured under different conditions {e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, Ka, Ka) are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.
The present antibodies or antigen-binding portions thereof have in vitro and in vivo therapeutic, prophylactic, and/or diagnostic utilities. For example, these antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to a subject, e.g., in vivo, to treat, inhibit, prevent relapse, and/or diagnose cancer.
Purified antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.
Where necessary, antibodies are analyzed for their biological activity. In some embodiments, antibodies of the invention are tested for their antigen binding activity. The antigen binding assays that are known in the art and can be used herein include without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, chemiluminescent immunoassays, nanoparticle immunoassays, aptamer immunoassays, and protein A immunoassays.
Uses
An antibody of the invention may be used in, for example, in vitro, ex vivo and in vivo therapeutic methods. Antibodies of the invention can be used as an antagonist to partially or fully block the specific antigen activity in vitro, ex vivo and/or in vivo. Moreover, at least some of the antibodies of the invention can neutralize antigen activity from other species. Accordingly, antibodies of the invention can be used to inhibit a specific antigen activity, e.g., in a cell culture containing the antigen, in human subjects or in other mammalian subjects having the antigen with which an antibody of the invention cross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse). In one embodiment, an antibody of the invention can be used for inhibiting antigen activities by contacting the antibody with the antigen such that antigen activity is inhibited. In one embodiment, the antigen is a human protein molecule.
“Administering” is referred to herein as providing a therapeutic composition of the invention to a patient. By way of example and not limitation, composition administration, e.g., injection, may be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes may be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route or nasal route. Additionally, administration may also be by surgical deposition of a bolus or positioning of a medical device.
In one embodiment, an antibody of the invention can be used in a method for inhibiting an antigen in a subject suffering from a disorder in which the antigen activity is detrimental, comprising administering to the subject an antibody of the invention such that the antigen activity in the subject is inhibited. In one embodiment, the antigen is a human protein molecule and the subject is a human subject. Alternatively, the subject can be a mammal expressing the antigen with which an antibody of the invention binds. Still further the subject can be a mammal into which the antigen has been introduced (e.g., by administration of the antigen or by expression of an antigen transgene). An antibody of the invention can be administered to a human subject for therapeutic purposes. Moreover, an antibody of the invention can be administered to a non-human mammal expressing an antigen with which the antibody cross-reacts (e.g., a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and time courses of administration). Antibodies of the invention can be used to treat, inhibit, delay progression of, prevent/delay recurrence of, ameliorate, or prevent diseases, disorders or conditions associated with abnormal expression and/or activity of Globo series antigens (Globo H, SSEA-3, SSEA-4), including but not limited to cancer, muscular disorders, ubiquitin-pathway-related genetic disorders, immune/inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders.
In certain embodiments, an immunoconjugate comprising an antibody of the invention conjugated with a cytotoxic agent is administered to the patient. In some embodiments, the immunoconjugate and/or antigen to which it is bound is/are internalized by cells expressing one or more proteins on their cell surface which are associated with Globo series antigens, resulting in increased therapeutic efficacy of the immunoconjugate in killing the target cell with which it is associated. In one embodiment, the cytotoxic agent targets or interferes with nucleic acid in the target cell. Examples of such cytotoxic agents include any of the chemotherapeutic agents noted herein (such as a maytansinoid or a calicheamicin), a radioactive isotope, or a ribonuclease or a DNA endonuclease.
Antibodies of the invention can be used either alone or in combination with other compositions in a therapy. For instance, an antibody of the invention may be co-administered with another antibody, and/or adjuvant/therapeutic agents (e.g., steroids). For instance, an antibody of the invention may be combined with an anti-inflammatory and/or antiseptic in a treatment scheme, e.g. in treating any of the diseases described herein, including cancer, muscular disorders, ubiquitin-pathway-related genetic disorders, immune/inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders. Such combined therapies noted above include combined administration (where the two or more agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, and/or following, administration of the adjunct therapy or therapies.
An antibody of the invention (and adjunct therapeutic agent) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
Therapeutic Applications
Described herein are therapeutic methods that include administering to a subject in need of such treatment a therapeutically effective amount of a composition that includes one or more antibodies described herein.
In some embodiments, the subject (e.g., a human patient) in need of the treatment is diagnosed with, suspected of having, or at risk for cancer. Examples of the cancer include, but are not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, oral cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, pancreas cancer, colon cancer, kidney cancer, cervix cancer, ovary cancer and prostate cancer. In some embodiments, the cancer is sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, or pancreas cancer. In some preferred embodiments, the cancer is brain cancer or glioblastoma multiforme (GBM) cancer.
In preferred embodiments, the antibody is capable of targeting Globo series antigens-expressing cancer cells. In some embodiments, the antibody is capable of targeting Globo series antigens on cancer cells. In some embodiments, the antibody is capable of targeting Globo series antigens in cancers.
The treatment results in reduction of tumor size, elimination of malignant cells, prevention of metastasis, prevention of relapse, reduction or killing of disseminated cancer, prolongation of survival and/or prolongation of time to tumor cancer progression.
In some embodiments, the treatment further comprises administering an additional therapy to said subject prior to, during or subsequent to said administering of the antibodies. In some embodiments, the additional therapy is treatment with a chemotherapeutic agent. In some embodiments, the additional therapy is radiation therapy.
The methods of the invention are particularly advantageous in treating and preventing early stage tumors, thereby preventing progression to the more advanced stages resulting in a reduction in the morbidity and mortality associated with advanced cancer. The methods of the invention are also advantageous in preventing the recurrence of a tumor or the regrowth of a tumor, for example, a dormant tumor that persists after removal of the primary tumor, or in reducing or preventing the occurrence of a tumor.
In some embodiments, the methods as disclosed herein are useful for the treatment or prevention of a cancer, for example where a cancer is characterized by increased Globo H, SSEA-3 and/or SSEA-4 expression. In some embodiments the cancer comprises a cancer stem cell. In some embodiments, the cancer is a pre-cancer, and/or a malignant cancer and/or a therapy resistant cancer. In some embodiments, the cancer is a brain cancer.
The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having cancer, which include, but not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, oral cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, pancreas cancer, colon cancer, kidney cancer, cervix cancer, ovary cancer and prostate cancer. A subject having cancer can be identified by routine medical examination.
“An effective amount” as used herein refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has cancer, a symptom of cancer, or a predisposition toward cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect cancer, the symptom of cancer, or the predisposition toward cancer.
“Development” or “progression” of cancer means initial manifestations and/or ensuing progression of cancer. Development of cancer can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of cancer includes initial onset and/or recurrence.
Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.
The exemplary therapeutic compositions (also referred to herein as pharmaceutical compositions) generally include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intramuscular, intra-arterial, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, phosphate buffered saline, tris-buffered saline, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH value can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
Exemplary pharmaceutical compositions suitable for an injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Exemplary Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Exemplary oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
Furthermore, for oral administration, the exemplary formulations of the invention can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. The compositions of the invention can be also introduced in microspheres or microcapsules, e.g., fabricated from poly-glycolic acid/lactic acid (PGLA) (see, U.S. Pat. Nos. 5,814,344; 5,100,669 and 4,849,222; PCT Publication Nos. WO 95/11010 and WO 93/07861). Liquid preparations for oral administration can take the form of, for example, solutions, syrups, emulsions or suspensions, or they can be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
For administration by inhalation, or nasal administration the exemplary compounds/formulations can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be transmucosal or transdermal. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
According to implementations, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811, which is incorporated by reference herein.
It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
The pharmaceutical formulations of the invention can be delivered parenterally, i.e., by intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.), subdermal (s.d.), or intradermal (i.d.) administration, by direct injection, via, for example, bolus injection, continuous infusion, or gene gun (e.g., to administer a vector vaccine to a subject, such as naked DNA or RNA). Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as excipients, suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
Dosage: Toxicity and therapeutic efficacy of such therapeutic compositions may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapeutic compositions which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected location to minimize potential damage to uninfected cells and, thereby, reduce side effects.
Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In some embodiments, a therapeutically effective amount of a therapeutic composition (i.e., an effective dosage) may range from about 0.001 μg/kg to about 250 g/kg, 0.01 μg/kg to 10 g/kg, or 0.1 μg/kg to 1.0 g/kg or about or at least: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009; 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09; 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, or 250 grams or micrograms per kilogram of patient body weight, or any range between any of the numbers listed herein, or other ranges that would be apparent and understood by artisans without undue experimentation. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.
In other embodiments, a therapeutically effective amount of Globo series moiety in the therapeutic composition (i.e., an effective dosage) may range from about 0.001 μg/kg to about 250 g/kg, 0.01 μg/kg to 10 g/kg, or 0.1 μg/kg to 1.0 g/kg or about or at least: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009; 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09; 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, or 250 grams or micrograms per kilogram of patient body weight, or any range between any of the numbers listed herein, or other ranges that would be apparent and understood by artisans without undue experimentation. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. In one embodiment, the immunogenically effective amount of a pharmaceutically acceptable carrier comprising the vaccine ranges from about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75 to about 5.0 μg, or any range between any of the numbers listed herein.
In some embodiments, the therapeutic compositions of the invention are administered to a subject in need thereof (e.g., one having a cancer such as breast cancer) in a method that on average extends progression free survival or overall survival over a control placebo, e.g., a phosphate buffered saline placebo, by about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 days, weeks, months, or years.
Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
In order to generate anti-Globo series antigens human monoclonal antibodies, B cells were isolated from peripheral blood of vaccinated patients. After administering Globo H-KLH vaccine (OBI-822/OBI-821) in a patient of recurrent ovarian cancer, blood samples were collected for the following analyzing procedure.
IgD−IgM−IgA− memory B cells were freshly isolated from human peripheral blood and plated into 384-well tissue culture plates at a density of one cell per well using fluorescence-activated cell sorter. The sorted B cells were stimulated to secrete IgG and incubated for several days. After incubation, B cell lysates and culture supernatants were collected separately.
In obtaining Globo H, SSEA-3 or SSEA-4 binding clones, genes encoding Ig VH, Ig Vκ or Ig Vλ are recovered from B cell lysates using RT-PCR and cloned into IgG expression vectors. Recombinant antibodies are expressed by transfection of mammalian cells and used to confirm the binding specificities or to implement other functional assays.
To screen for anti-Globo series antigen human antibodies, the culture supernatants containing secreted IgG were assayed for Globo H, SSEA-3 or SSEA-4 binding specificities using ELISA.
1. Reagent/Buffer Preparation:
A. Globo H-ceramide, Globo H-lipid, SSEA-3-ceramide, SSEA-3-lipid, SSEA-4-ceramide and SSEA-4-lipid powder was dissolved in ethanol and storage at −20° C. 20 μg of antigen was added into 5 mL ethanol and mixed gently.
B. Adding 50 μL of coating antigen (0.2 μg of antigen/well) into each well. Covering, labeling and incubating at room temperature for overnight.
C. Adding 100 μL/well of Blocking Buffer (Sigma, Cat #B6429) into each well and incubating at room temperature for 30 minutes.
2. Addition of Culture Supernatants to Antigen-Coated Plate
A. After the blocking procedure, washing three times with 200 μL PBST Wash Buffer.
B. Transferring 50 μL of all diluted test samples to corresponding wells in the Antigen-Coated/Blocked Plate.
C. Incubating the plate at room temperature for 1 hour.
D. After incubating, washing three times with 200 μL PBST Wash Buffer.
3. Addition of Secondary Antibody to Antigen-Coated Plate
A. Pipetting 25 μL of Secondary Antibody to 4975 μL of Blocking Buffer and mixing gently. (Goat anti-human IgG-AP for IgG antibody detection)
B. Adding 50 μL of Secondary Antibody Solution and incubating at room temperature for 45 minutes.
C. After incubating, washing four times with 200 μL Wash Buffer.
D. Adding 100 μL Substrate Solution (Sigma, Cat #P7998) and incubating for 20 minutes at 37° C.
E. Stop reaction by adding 50 μL of Stop Solution (Sigma, Cat #A5852), mixing well and then reading the absorbance at 405 nm on the ELISA Plate Reader.
4. Data Analysis
A. The well that gives a reading above the cutoff value is defined as the potential Globo series antigen binding clone.
B. Cutoff value=X+0.1. (X is the mean OD value of negative control).
C. Controls were treated the same as test samples. The differences are that the positive control has primary Abs known positive Ab (anti-Globo H, anti-SSEA-3, anti-SSEA-4 antibody or no IgG added as the primary antibody is negative control).
D. Data were analyzed statistically by Mann-Whitney test using GraphPad Prism 5 Software.
5. Result
Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.
All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.
This application claims the priority to and benefit of International Patent Application No. PCT/US2017/044713, filed Jul. 27, 2017, which claims priorty to and benefit of U.S. Provisional Patent Application No. 62/368,407, filed Jul. 29, 2016. The entirety of the aforementioned applications are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/044713 | 7/31/2017 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/023121 | 2/1/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 3773919 | Boswell et al. | Nov 1973 | A |
| 4203975 | Greven | May 1980 | A |
| RE30985 | Cartaya | Jun 1982 | E |
| 4419446 | Howley et al. | Dec 1983 | A |
| 4522811 | Eppstein et al. | Jun 1985 | A |
| 4560655 | Baker | Dec 1985 | A |
| 4601903 | Frasch | Jul 1986 | A |
| 4601978 | Karin | Jul 1986 | A |
| 4657866 | Kumar | Apr 1987 | A |
| 4676980 | Segal et al. | Jun 1987 | A |
| 4767704 | Cleveland et al. | Aug 1988 | A |
| 4816567 | Cabilly et al. | Mar 1989 | A |
| 4849222 | Broaddus | Jul 1989 | A |
| 4927762 | Darfler | May 1990 | A |
| 4943533 | Mendelsohn et al. | Jul 1990 | A |
| 4965199 | Capon et al. | Oct 1990 | A |
| 4975278 | Senter et al. | Dec 1990 | A |
| 5004697 | Pardridge | Apr 1991 | A |
| 5057540 | Kensil et al. | Oct 1991 | A |
| 5061620 | Tsukamoto et al. | Oct 1991 | A |
| 5100669 | Hyon et al. | Mar 1992 | A |
| 5112596 | Malfroy-Camine | May 1992 | A |
| 5122469 | Mather et al. | Jun 1992 | A |
| 5212290 | Vogelstein et al. | May 1993 | A |
| 5264365 | Georgiou et al. | Nov 1993 | A |
| 5268164 | Kozarich et al. | Dec 1993 | A |
| 5500362 | Robinson et al. | Mar 1996 | A |
| 5506206 | Kozarich et al. | Apr 1996 | A |
| 5508192 | Georgiou et al. | Apr 1996 | A |
| 5545806 | Lonberg et al. | Aug 1996 | A |
| 5545807 | Surani et al. | Aug 1996 | A |
| 5565332 | Hoogenboom et al. | Oct 1996 | A |
| 5569825 | Lonberg et al. | Oct 1996 | A |
| 5571894 | Wels et al. | Nov 1996 | A |
| 5580717 | Dower et al. | Dec 1996 | A |
| 5587458 | King et al. | Dec 1996 | A |
| 5625126 | Lonberg et al. | Apr 1997 | A |
| 5633425 | Lonberg et al. | May 1997 | A |
| 5641870 | Rinderknecht et al. | Jun 1997 | A |
| 5648237 | Carter | Jul 1997 | A |
| 5661016 | Lonberg et al. | Aug 1997 | A |
| 5686416 | Kozarich et al. | Nov 1997 | A |
| 5733743 | Johnson et al. | Mar 1998 | A |
| 5804396 | Plowman | Sep 1998 | A |
| 5814344 | Tice et al. | Sep 1998 | A |
| 5821337 | Carter et al. | Oct 1998 | A |
| 5840523 | Simmons et al. | Nov 1998 | A |
| 5869046 | Presta et al. | Feb 1999 | A |
| 5891996 | Mateo de Acosta del Rio et al. | Apr 1999 | A |
| 6004940 | Marasco et al. | Dec 1999 | A |
| 6027888 | Georgiou et al. | Feb 2000 | A |
| 6083715 | Georgiou et al. | Jul 2000 | A |
| 6265150 | Terstappen et al. | Jul 2001 | B1 |
| 6329173 | Marasco et al. | Dec 2001 | B1 |
| 6524584 | Kensil | Feb 2003 | B2 |
| 6544952 | Danishefsky et al. | Apr 2003 | B1 |
| 6703019 | Malfroy-Camine | Mar 2004 | B1 |
| 7595292 | Brocchini et al. | Sep 2009 | B2 |
| 8268969 | Wong et al. | Sep 2012 | B2 |
| 9850473 | Wang | Dec 2017 | B2 |
| 10815307 | Yu et al. | Oct 2020 | B2 |
| 20020025313 | Micklus et al. | Feb 2002 | A1 |
| 20020038086 | Hynynen et al. | Mar 2002 | A1 |
| 20020065259 | Schatzberg et al. | May 2002 | A1 |
| 20030073713 | Schoenhard | Apr 2003 | A1 |
| 20030083299 | Ferguson | May 2003 | A1 |
| 20030104402 | Zauderer et al. | Jun 2003 | A1 |
| 20030129186 | Beliveau et al. | Jul 2003 | A1 |
| 20030153492 | Danishefsky et al. | Aug 2003 | A1 |
| 20030162695 | Schatzberg et al. | Aug 2003 | A1 |
| 20040018194 | Francisco et al. | Jan 2004 | A1 |
| 20040131692 | Kreuter et al. | Jul 2004 | A1 |
| 20040204354 | Nelson et al. | Oct 2004 | A1 |
| 20040208884 | Danishefsky et al. | Oct 2004 | A1 |
| 20040229310 | Simmons | Nov 2004 | A1 |
| 20040247608 | Krantz et al. | Dec 2004 | A1 |
| 20050048572 | Reilly et al. | Mar 2005 | A1 |
| 20050089473 | Black et al. | Apr 2005 | A1 |
| 20050124533 | Schatzberg et al. | Jun 2005 | A1 |
| 20060035267 | Livingston et al. | Feb 2006 | A1 |
| 20070059769 | Blixt et al. | Mar 2007 | A1 |
| 20090317411 | Wong et al. | Dec 2009 | A1 |
| 20100136042 | Wong | Jun 2010 | A1 |
| 20100166790 | Agadjanyan et al. | Jul 2010 | A1 |
| 20100286035 | Ohtaki et al. | Nov 2010 | A1 |
| 20110117009 | Kratz et al. | May 2011 | A1 |
| 20120237532 | Olbrich et al. | Sep 2012 | A1 |
| 20120294859 | Goletz et al. | Nov 2012 | A1 |
| 20120321583 | Yurkovetskiy et al. | Dec 2012 | A1 |
| 20120328646 | Wong et al. | Dec 2012 | A1 |
| 20130095173 | Danishefsky et al. | Apr 2013 | A1 |
| 20130232589 | Papkoff et al. | Sep 2013 | A1 |
| 20140363455 | Stull et al. | Dec 2014 | A1 |
| 20150030669 | Platscher et al. | Jan 2015 | A1 |
| 20150087814 | Wang et al. | Mar 2015 | A1 |
| 20150297696 | Yu et al. | Oct 2015 | A1 |
| 20150316556 | Hardt et al. | Nov 2015 | A1 |
| 20150344551 | Wong et al. | Dec 2015 | A1 |
| 20160051672 | Stewart et al. | Feb 2016 | A1 |
| 20160074522 | Okuda et al. | Mar 2016 | A1 |
| 20160102151 | Wong et al. | Apr 2016 | A1 |
| 20160339089 | Yu et al. | Nov 2016 | A1 |
| 20170067885 | Yu et al. | Mar 2017 | A1 |
| 20170101462 | Yu et al. | Apr 2017 | A1 |
| 20170283488 | Yu et al. | Oct 2017 | A1 |
| 20170283489 | Bosio et al. | Oct 2017 | A1 |
| 20170304419 | Yu et al. | Oct 2017 | A1 |
| 20170335281 | Loew et al. | Nov 2017 | A1 |
| 20180028629 | Yu et al. | Feb 2018 | A1 |
| 20180193481 | Chang et al. | Jul 2018 | A1 |
| 20180208915 | Kawaguchi et al. | Jul 2018 | A1 |
| 20180291109 | Lin et al. | Oct 2018 | A1 |
| 20180339061 | Yu et al. | Nov 2018 | A1 |
| Number | Date | Country |
|---|---|---|
| 1871025 | Nov 2006 | CN |
| 103108654 | May 2013 | CN |
| 0404097 | Dec 1990 | EP |
| 1391213 | Feb 2004 | EP |
| 2993182 | Mar 2016 | EP |
| 2006-507233 | Mar 2006 | JP |
| 2011524375 | Sep 2011 | JP |
| 2011524417 | Sep 2011 | JP |
| 2016500256 | Jan 2016 | JP |
| 10-2012-0014238 | Feb 2012 | KR |
| WO 8700195 | Jan 1987 | WO |
| WO 9003184 | Apr 1990 | WO |
| WO 9003430 | Apr 1990 | WO |
| WO 9100360 | Jan 1991 | WO |
| WO 9110741 | Jul 1991 | WO |
| WO 9200373 | Jan 1992 | WO |
| WO 9209690 | Jun 1992 | WO |
| WO 9301161 | Jan 1993 | WO |
| WO 9306213 | Apr 1993 | WO |
| WO 93007861 | Apr 1993 | WO |
| WO 9308829 | May 1993 | WO |
| WO 9316185 | Aug 1993 | WO |
| WO 9404690 | Mar 1994 | WO |
| WO 94011026 | May 1994 | WO |
| WO 95011010 | Apr 1995 | WO |
| WO 9607754 | Mar 1996 | WO |
| WO 9611711 | Apr 1996 | WO |
| WO 9630347 | Oct 1996 | WO |
| WO 9633735 | Oct 1996 | WO |
| WO 9633978 | Oct 1996 | WO |
| WO 9633980 | Oct 1996 | WO |
| WO 9634096 | Oct 1996 | WO |
| WO 9640210 | Dec 1996 | WO |
| WO 9738983 | Oct 1997 | WO |
| WO 9824893 | Jun 1998 | WO |
| WO 9836772 | Aug 1998 | WO |
| WO 9843960 | Oct 1998 | WO |
| WO 9906378 | Feb 1999 | WO |
| WO 9906396 | Feb 1999 | WO |
| WO 9909016 | Feb 1999 | WO |
| WO 99042130 | Aug 1999 | WO |
| WO 200041720 | Jul 2000 | WO |
| WO 200048630 | Aug 2000 | WO |
| WO-200049412 | Aug 2000 | WO |
| WO 2003015796 | Feb 2003 | WO |
| WO 2003043583 | May 2003 | WO |
| WO 2003077945 | Sep 2003 | WO |
| WO 2004011476 | Feb 2004 | WO |
| WO 2004032828 | Apr 2004 | WO |
| WO 2005007197 | Jan 2005 | WO |
| WO 2006105152 | Oct 2006 | WO |
| WO 2006134423 | Dec 2006 | WO |
| WO 2007026190 | Mar 2007 | WO |
| 2007047764 | Apr 2007 | WO |
| WO 2007044515 | Apr 2007 | WO |
| WO 2009035494 | Mar 2009 | WO |
| WO 2009126737 | Oct 2009 | WO |
| 2010005735 | Mar 2010 | WO |
| WO-2011156774 | Dec 2011 | WO |
| WO 2014107652 | Jul 2014 | WO |
| WO 2014178195 | Nov 2014 | WO |
| WO 2015143123 | Sep 2015 | WO |
| WO 2015157629 | Oct 2015 | WO |
| WO 2015159118 | Oct 2015 | WO |
| 2015157629 | Dec 2015 | WO |
| WO 2016026742 | Feb 2016 | WO |
| WO 2016044326 | Mar 2016 | WO |
| 2016118961 | Jul 2016 | WO |
| WO 2016118961 | Jul 2016 | WO |
| WO 2016123593 | Aug 2016 | WO |
| 2017004150 | Jan 2017 | WO |
| WO 2017041027 | Mar 2017 | WO |
| 2017062792 | Apr 2017 | WO |
| WO 2017062792 | Apr 2017 | WO |
| 2016044326 | May 2017 | WO |
| 2017185089 | Oct 2017 | WO |
| WO 2017172990 | Oct 2017 | WO |
| WO 2018002640 | Jan 2018 | WO |
| 2018022933 | Feb 2018 | WO |
| WO 2018022933 | Feb 2018 | WO |
| WO 2018023121 | Feb 2018 | WO |
| WO 2018094414 | May 2018 | WO |
| Entry |
|---|
| Mariuzza (Annu. Rev. Biophys. Biophys. Chem., 16: 139-159, 1987). |
| McCarthy et al. (J. Immunol. Methods, 251(1-2): 137-149, 2001). |
| Lin et al. (African Journal of Biotechnology, 10(79):18294-18302, 2011). |
| Büll, Christian, et al. “Sialic acid blockade suppresses tumor growth by enhancing T-cell-mediated tumor immunity.” Cancer Research 78.13 (2018): 3574-3588. |
| Chuang, Po-Kai, et al. “Signaling pathway of globo-series glycosphingolipids and β1, 3-galactosyltransferase V (β3GalT5) in breast cancer.” Proceedings of the National Academy of Sciences 116.9 (2019): 3518-3523. |
| International Search Report/Written Opinion dated Oct. 18, 2019 in counterpart application PCT/US2019/035168, 13 pages. |
| International Search Report dated Dec. 3, 2019 in counterpart application PCT/US2019/039414, 5 pages. |
| Ragupathi, Govindaswami, et al. “A novel and efficient method for synthetic carbohydrate conjugate vaccine preparation: synthesis of sialyl Tn-KLH conjugate using a 4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazide (MMCCH) linker arm.” Glycoconjugate Journal 15.3 (1998): 217-221. |
| Final Office Action issued in U.S. Appl. No. 16/454,750 dated May 27, 2021. |
| Substantive Examination Report, Office Paper No. 7, issued in Philippines Application No. 1-2017-500478 dated Oct. 21, 2020. |
| Substantive Examination Report, Office Paper No. 9, issued in Philippines Application No. 1-2017-500478 dated Jun. 10, 2021. |
| Abrahmsén et al, “Analysis of signals for secretion in the staphylococcal protein A gene,” EMBO J., Dec. 30, 1985, 4(13B):3901-3906. |
| Allen, P. Z. et al., Immunochemical Studies on a Sophorosyl-Azoprotein Conjugate, Biochemistry, 1967, 6(10), 3029-3036. |
| Arié et al., “Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli,” Mol. Microbiol., Jan. 2001, 39(1):199-210. |
| Arigi, Emma, et al. “Design of a covalently bonded glycosphingolipid microarray.” Glycoconjugate Journal 29.1 (2012): 1-12. |
| Avery, Oswald et al., Chemo-Immunological Studies on Conjugated Carbohydrate-Proteins, J. Exp. Med., 1929, 50, 533-550. |
| Bachmann, Cellular and Molecular Biology, vol. 2, Chapter 72: Derivations and Genotypes of Some Mutant Derivatives of Escherichia coli K-12, Neidhardt et al., eds., 1987, pp. 1190-1219, American Society for Microbiology, Washington, D.C. |
| Baldwin et al., “Monoclonal antibodies in cancer treatment,” Lancet, Mar. 15, 1986, 327(8481):603-605. |
| Barbas et al., “Assembly of combinatorial antibody libraries on phage surfaces: the gene III site,” Proc. Natl. Acad. Sci. U.S.A., Sep. 15, 1991, 88(18):7978-7982. |
| Barbas, C.F. et al., “Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem” Proc. Natl. Acad. Sci. USA, May 15, 1992, 89(10): 4457-4461. |
| Barbas et al., “In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity,” Proc. Nat. Acad. Sci. U.S.A., Apr. 26, 1994, 91(9):3809-3813. |
| Barnes et al., “Methods for growth of cultured cells in serum-free medium,” Anal. Biochem., Mar. 1, 1980, 102(2):255-270. |
| Bass et al., “Hormone phage: an enrichment method for variant proteins with altered binding properties,” Proteins, 1990, 8(4):309-314. |
| Berenbaum, M. C., What is Synergy?, Pharmacol. Rev. 41(2) :93-141, 1989. |
| Bergman, Jan, and Lennart Venemalm. “Efficient synthesis of 2-chloro-, 2-bromo-, and 2-iodoindole.” The Journal of Organic Chemistry 57.8 (1992): 2495-2497. |
| Bertozzi, CR et al., Glycans in Cancer and Inflammation—Potential for Therapeutics and Diagnostics, Nat Rev Drug Discovery, 2005, 4, 477-488. |
| Bhaskar, Vinay, et al. “E-selectin up-regulation allows for targeted drug delivery in prostate cancer.” Cancer Research 63.19 (2003): 6387-6394. |
| Bird, R.E., et al., “Single-chain antigen-binding proteins” Science Oct. 21, 1988; 242(4877):423-426. |
| Bliss, C.I., The Calculation of Microbial Assays, Bacterial. Rev. 20:243-258, 1956. |
| Bobo et al., “Convection-enhanced delivery of macromolecules in the brain,” Proc. Natl. Acad. Sci. U.S.A., Mar. 15, 1994, 91(6) 2076-2080. |
| Boerner et al., “Production of antigen-specific human monoclonal antibodies from in vitro-primed human splenocytes,” J. Immunol., Jul. 1, 1991, 147(1):86-95. |
| Borisy, Alexis et al., Systematic Discovery of Multicomponent Therapeutics, Proc. Natl. Acad. Sci. 100(13):7977-7982, 2003. |
| Bosse, Folkert et al., Linear Synthesis of the Tumor-Associated Carbohydrate Antigens Globo-H, SSEA-3, and Gb3, J Org Chem. 67(19):6659-70, 2002. |
| Bothmann et al., “The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis-prolines,” J. Biol. Chem., Jun. 2, 2000, 275(22):17100-17105. |
| Bowie, Ju et al., Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions, Science, 247: 1306-1310 (1990). |
| Bremer, E. G., et al. “Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland.” Journal of Biological Chemistry 259.23 (1984): 14773-14777. |
| Brennan et al., “Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments,” Science, Jul. 5, 1985, 229(4708):81-83. |
| Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Chapter 4: Mouse-Human Myeloma Partners for the Production of Heterohybridomas, Schook, ed., 1987, pp. 51-63, Marcel Dekker, Inc., New York. |
| Brüggemann et al., “Designer mice: the production of human antibody repertoires in transgenic animals,” Year in Immunol., 1993, 7:33-40. |
| Carter et al., “High level Escherichia coli expression and production of a bivalent humanized antibody fragment,” Nature Biotechnology, Feb. 1992, 10(2):163-167. |
| Carter et al., “Humanization of an anti-p185HER2 antibody for human cancer therapy,” Proc. Natl. Acad. Sci. U.S.A., May 15, 1992, 89(10):4285-4289. |
| Casset, Florence, et al. “A peptide mimetic of an anti-CD4 monoclonal antibody by rational design.” Biochemical and Biophysical Research Communications 307.1 (2003): 198-205. |
| Chang et al., “Potent immune-modulating and anticancer effects of NKT cell stimulatory glycolipids,” Proc. Natl. Acad. Sci. USA, Jun. 19, 2007, 104(25):10299-10304. |
| Chang et al., “Expression of Globo H and SSEA3 in breast cancer stem cells and the involvement of fucosyl transferases 1 and 2 in Globo H synthesis,” Proc. Natl. Acad. Sci. U.S.A., Aug. 19, 2008, 105(33):11667-11672. |
| Chen et al., “Chaperone activity of DsbC,” J. Bio. Chem., July 9. 1999, 274(28):19601-19605. |
| Chen et al., “Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen,” J. Mol. Biol., Nov. 5, 1999, 293(4):865-881. |
| Chen et al., “Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists,” Proc. Natl. Acad. Sci. U.S.A., Apr. 13, 1999, 96(8):4325-4329. |
| Chen, Wei, et al. “Determination of thiols and disulfides via HPLC quantification of 5-thio-2-nitrobenzoic acid.” Journal of Pharmaceutical and Biomedical Analysis 48.5 (2008): 1375-1380. |
| Cheung, Sarah et al., Stage-Specific Embryonic Antigen-3 (SSEA-3) and β3GalT5 are cancer specific and Significant Markers for Breast Cancer Stem Cells, PNAS, Jan. 26, 2016, vol. 113, No. 4, pp. 960-965. |
| Chothia et al., “Canonical structures for the hypervariable regions of immunoglobulins,” J. Mol. Biol., Aug. 20, 1987, 196(4):901-917. |
| Chou, Ting-Chao and Talalay, Paul, A Simple Generalized Equation for the Analysis of Multiple Inhibitions of Michaelis-Menten Kinetic Systems, J. Biol. Chem. 252:6438-6442, 1977. |
| Chou, T. C. and Talalay, P., Quantitative Analysis of Dose-Effect Relationships: The Combined Effects of Multiple Drugs or Enzyme Inhibitors. Adv. Enzyme Regul. 22:27-55, 1984. |
| Clackson et al., “Making antibody fragments using phage display libraries,” Nature, Aug. 15, 1991, 352(6336):624-628. |
| ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Jan. 24, 2012—Trial of Active Imunotherapy with Globo H-KLH (OPT-822) in Metastatic Breast Cancer Subjects); Jan. 24, 2012 [cited Oct. 11, 2017]; [about 7 screens]. Available from: https:clinicaltrials.gov/ct2/show/NCT01516307. |
| Clynes, Raphael, et al. “Fc receptors are required in passive and active immunity to melanoma.” Proceedings of the National Academy of Sciences 95.2 (1998): 652-656. |
| Colman, P.M., “Effects of amino acid sequence changes on antibody-antigen interactions,” Research in Immunology 145:33-36, 1994. |
| Cunningham et al., “High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis,” Science, Jun. 2, 1989, 244(4908):1081-1085. |
| Cuzick, J., et al. “Overview of the main outcomes in breast-cancer prevention trials.” The Lancet 361.9354 (2003): 296-300. |
| Danishefsky, Samuel J., et al. “Development of Globo-H cancer vaccine.” Accounts of Chemical Research 48.3 (2015): 643-652. |
| De Pascalis, Roberto, et al. “Grafting of “abbreviated” complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody.” The Journal of Immunology 169.6 (2002): 3076-3084. |
| Doronina, Svetlana O., et al. “Development of potent monoclonal antibody auristatin conjugates for cancer therapy.” Nature Biotechnology 21.7 (2003): 778-784. |
| Eller, Chelcie et al., Human Cancer Antigen Globo H Is a Cell-Surface Ligand for Human Ribonuclease 1, ACS Central Science. vol. 1, p. 181-90, Jul. 13, 2015. |
| Embleton et al., “In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells,” Nucl. Acids Res., Aug. 11, 1992, 20(15):3831-3837. |
| Engels et al., “Gene synthesis [new synthetic methods (77)],” Angew. Chem. Int. Ed. Engl., Jun. 1989, 28(6):716-734. |
| Evans, T. R. J., and S. B. Kaye. “Vaccine therapy for cancer—fact or fiction?” Q J Med 92.6 (1999): 299-307. |
| Extended European Search Report, Application No. 15842660.1, dated Mar. 12, 2018, 9 pages. |
| Extended European Search Report from corresponding European App. No. 16843131.0, dated Feb. 14, 2019, 13 Pages. |
| Fellouse et al., “Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition,” Proc. Natl. Acad. Sci. U.S.A., Aug. 24, 2004, 101(34):12467-12472. |
| Feng, Li. “Probing lipid-protein interactions using lipid microarrays.” Prostaglandins & other lipid mediators 77.1-4 (2005): 158-167. |
| Fielder, R. J. et al., An Immunogenic Polysaccharide-Protein Conjugate, J. Immunol., 1970, 105(1), 265-267. |
| Fishwild et al., “High-avidity human IgGk monoclonal antibodies from a novel strain of minilocus transgenic mice,” Nature Biotechnol., Jul. 1996, 14(7):845-851. |
| Fitzgerald, Jonathan et al., Systems Biology and Combination Therapy in the Quest for Clinical Efficacy, Nature Chem. Biol. 2(9):458-466, 2006. |
| Francisco, Joseph A., et al. “cAC10-vcMMAE, an anti-CD30—monomethyl auristatin E conjugate with potent and selective antitumor activity.” Blood 102.4 (2003): 1458-1465. |
| Galfrè et al., “Preparation of monoclonal antibodies: strategies and procedures,” Methods Enzymol., 1981, 73(Pt B):3-46. |
| Gazzano-Santoro, Hélène, et al. “A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody.” Journal of Immunological Methods 202.2 (1997): 163-171. |
| Gijsen, H.J. et al., Recent Advances in the Chemoenzymatic Synthesis of Carbohydrates and Carbohydrate Mimetics, Chem. Rev., 96, 443-473, 1996. |
| Gilewski, Teresa et al., Immunization of Metastatic Breast Cancer Patients with a Fully Synthetic Globo H Conjugate: A Phase I Trial, Proc Natl Acad Sci USA 98:3270-3275, 2001. |
| Gill et al., “Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease,” Nature Med., May 2003, 9(5):589-595 and Addendum from Apr. 2006, 12(4):479. |
| Goding, Monoclonal Antibodies: Principles and Practice 2nd ed., Chapter 3: Production of Monoclonal Antibodies, 1986, pp. 59-103, Academic Press, London. |
| Goebel, Walther et al., Chemo-immunological Studies on Conjugated Carbohydrate-Proteins, J. Exp. Med., 1929, 50, 521-531. |
| Gonnet, GH et al., Exhaustive Matching of the Entire Protein Sequence Database, Science 256: 1443-1445 (1992). |
| Graham et al., “Characteristics of a human cell line transformed by DNA from human adenovirus type 5,” J. Gen. Virol., Jul. 1977, 36(1):59-72. |
| Gram et al., “In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library,” Proc. Natl. Acad. Sci. U.S.A., Apr. 15, 1992, 89(8):3576-3580. |
| Grant, Oliver C., et al. “Presentation, presentation, presentation! Molecular-level insight into linker effects on glycan array screening data.” Glycobiology 24.1 (2014): 17-25. |
| Greco, William et al., The Search for Synergy: A Critical Review From a Response Surface Perspective, Pharmacol. Rev. 47(2) :331-385, 1995. |
| Griffiths et al., “Human anti-self antibodies with high specificity from phage display libraries,” EMBO J., Feb. 1993, 12(2):725-734. |
| Gruber et al., “Efficient tumor cell lysis mediated by a bispecific single chain antibody expressed in Escherichia coli,” J. Immunol., Jun. 1, 1994, 152(11):5368-5374. |
| Guss et al., “Structure of the IgG-binding regions of streptococcal protein G,” EMBO J., Jul. 1986, 5(7):1567-1575. |
| Hakomori et al., “Glycosphingolipid antigens and cancer therapy,” Chem. & Biol., Feb. 1997, 4(2):97-104. |
| Hakomori, Sen-Itiroh, Tumor-associated carbohydrate antigens defining tumor malignancy: Basis for development of and-cancer vaccines, 2001, Advances in Experimental Medicine and Biology. 491 :369-402. |
| Ham, Richard et al., Media and Growth Requirements, Meth. Enz 58, 44-93 (1979). |
| Hammerling et al., “Production of antibody-producing hybridomas in the rodent systems.” in: Monoclonal Antibodies and T-Cell Hybridomas, 563-587, 1981, Elsevier North-Holland. |
| Hara et al., “Overproduction of penicillin-binding protein 7 suppresses thermosensitive growth defect at low osmolarity due to an spr mutation of Escherichia coli,” Microbial Drug Resistance, Spring 1996, 2(1):63-72. |
| Harris, “Production of humanized monoclonal antibodies for in vivo imaging and therapy,” Biochem. Soc. Transactions, Nov. 1995, 23(4):1035-1038. |
| Harris, J. Robin, et al. “Keyhole limpet hemocyanin (KLH), II: Characteristic reassociation properties of purified KLH1 and KLH2.” Micron 28.1 (1997): 43-56. |
| Harris, J. R., and J. Mark 1. “Keyhole limpet hemocyanin (KLH): a biomedical review.” Micron 30.6 (1999): 597-623. |
| Hawkins et al., “Selection of phage antibodies by binding affinity. Mimicking affinity maturation,” J. Mol. Biol., 1992, 226(3):889-896. |
| Heffernan, Michael J., et al. “In vivo efficacy of a chitosan/IL-12 adjuvant system for protein-based vaccines.” Biomaterials 32.3 (2011): 926-932. |
| Hernández-Ledesma, Blanca, Chia-Chien Hsieh, and O. Ben. “Lunasin, a novel seed peptide for cancer prevention.” Peptides 30.2 (2009): 426-430. |
| Himmelspach, K. et al., Use of 1-(m-aminophenyl)flavazoles for the Preparation of Immunogens with Oligosaccharide Determinant Groups, Eur. J. Immunol., 1971, 1(2), 106-112. |
| Hinman et al., “Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics,” Cancer Res., Jul. 15, 1993, 53(14):3336-3342. |
| Hirabayashi, J. et al., Oligosaccharide Microarrays for Glycomics, Trends in Biotechnology 21 (4): 141-143, 2003. |
| Hirano, Fumiya, et al. “Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity.” Cancer Research 65.3 (2005): 1089-1096. |
| Hogrefe, H.H. et al., “A bacteriophage lambda vector for the cloning and expression of immunoglobulin Fab fragments on the surface of filamentous phage” Gene, 1993, 128(1): 119-126. |
| Holliger et al., “‘Diabodies’: small bivalent and bispecific antibody fragments,” Proc. Natl. Acad. Sci. U.S.A., Jul. 15, 1993, 90(14):6444-6448. |
| Holm, Patrik, Rozbeh Jafari, and Birgitta E. Sundström. “Functional mapping and single chain construction of the anti-cytokeratin 8 monoclonal antibody TS1.” Molecular Immunology 44.6 (2007): 1075-1084. |
| Hoogenboom et al., “By-passing immunisation: Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro,” J. Mol. Biol., Sep. 20, 1992, 227(2):381-388. |
| Hoogenboom et al., “Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains,” Nucl. Acids Res., Aug. 11, 1991 19(15):4133-4137. |
| Huang, Cheng-Yuan et al., Carbohydrate Microarray for Profiling the Antibodies Interacting with Globo H Tumor Antigen, Proc Natl Acad Sci, 103:15-20, 2006. |
| Huang, Yen-Lin, and Chung-Yi Wu. “Carbohydrate-based vaccines: challenges and opportunities.” Expert Review of Vaccines 9.11 (2010): 1257-1274. |
| Huang, Yen-Lin, et al. “Carbohydrate-based vaccines with a glycolipid adjuvant for breast cancer.” Proceedings of the National Academy of Sciences 110.7 (2013): 2517-2522. |
| Hurle et al., “Protein engineering techniques for antibody humanization,” Curr. Opin. Biotechnol., Aug. 1994, 5(4):428-433. |
| Huston, James et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli” Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883. |
| International Search Report dated Jan. 8, 2016 in counterpart application PCT/IB2014/002744, 3 pages. |
| International Search Report and Written Opinion of the International Searching Authority, from corresponding International Patent Application No. PCT/US2015/050270, dated Dec. 15, 2015, 14 Pages. |
| International Search Report and Written Opinion of the International Searching Authority, from corresponding International Patent Application No. PCT/US16/50252, dated Nov. 17, 2016, 12 Pages. |
| International Search Report and Written Opinion dated Jul. 7, 2017, from corresponding International Patent Application No. PCT/US2017/024853, by Yu, Cheng-Der Tony et al., “Antibodies, Pharmaceutical Compositions and Methods”, filed Mar. 29, 2017, 21 pages. |
| International Search Report/Written Opinion dated Oct. 31, 2017 in counterpart PCT Application No. PCT/US2017/044244, 13 pages. |
| International Search Report dated Nov. 28, 2017 in counterpart application PCT/US2017/044713, 6 pages. |
| International Search Report/Written Opinion dated Mar. 12, 2018 in counterpart PCT Application No. PCT/US17/062886, 22 pages. |
| Jackson et al., “In vitro antibody maturation: Improvement of a high affinity, neutralizing antibody against IL-1β,” J. Immunol., Apr. 1, 1995, 154(7):3310-3319. |
| Jakobovits et al., “Analysis of homozygous mutant chimeric mice: deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production,” Proc. Natl. Acad. Sci. U.S.A., Mar. 15, 1993, 90(6):2551-2555. |
| Jakobovits et al., “Germ-line transmission and expression of a human-derived yeast artificial chromosome,” Nature, Mar. 18, 1993, 362(6417):255-258. |
| Jeon, Insik et al., A Practical Total Synthesis of Globo-H for Use in Anticancer Vaccines, J. Org. Chem., 2009, 74(21), pp. 8452-8455. |
| Jones et al., “Rapid PCR-cloning of full-length mouse immunoglobulin variable regions,” Nature Biotechnol., Jan. 1991, 9(1):88-89. |
| Jones et al., “Replacing the complementarity-determining regions in a human antibody with those from a mouse,” Nature, May 29-Jun. 4, 1986, 321(6069):522-525. |
| Jones, “Analysis of polypeptides and proteins,” Adv. Drug Delivery Rev., Jan.-Apr. 1993, 10(1):29-90. |
| Kam et al., “Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction,” Proc. Natl. Acad. Sci. U.S.A., Aug. 16, 2005, 102(33):11600-11605. |
| Kannagi, Reiji, et al. “Stage-specific embryonic antigens (SSEA-3 and-4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells.” EMBO Journal 2.12 (1983): 2355-2361. |
| Kannagi et al., “New globoseries glycosphingolipids in human teratocarcinoma reactive with the monoclonal antibody directed to a developmentally regulated antigen, stage-specific embryonic antigen 3,” J. Biol. Chem., Jul. 25, 1983, 258(14):8934-8942. |
| Klussman, Kerry, et al. “Secondary mAh-vcMMAE conjugates are highly sensitive reporters of antibody internalization via the lysosome pathway.” Bioconjugate chemistry 15.4 (2004): 765-773. |
| Koeller, Kathryn et al., Enzymes for Chemical Synthesis, Nature, 409, 232-240, 2001. |
| Köhler et al., “Continuous cultures of fused cells secreting antibody of predefined specificity,” Nature, Aug. 7, 1975, 256(5517):495-497. |
| Komenaka, Ian, Heidi Hoerig, and Howard L. Kaufman. “Immunotherapy for melanoma.” Clinics in Dermatology 22.3 (2004): 251-265. |
| Konecny, G. et al., Drug Interactions and Cytotoxic Effects of Paclitaxel in Combination with Carboplatin, Epirubicin, Gemcitabine or Vinorelbine in Breast Cancer Cell Lines and Tumor Samples, Breast Cancer Res. and Treatment 67:223-233, 2001. |
| Kontermann, “Intrabodies as therapeutic agents,” Methods, Oct. 2004, 34(2):163-170. |
| Kostelny et al., “Formation of a bispecific antibody by the use of leucine zippers,” J. Immunol., Mar. 1, 1992, 148(5):1547-1553. |
| Kozbor, “A human hybrid myeloma for production of human monoclonal antibodies,” J. Immunol., Dec. 1984, 133(6):3001-3005. |
| Krainer, Florian et al., An Updated View on Horseradish Peroxidases: Recombinant Production and Biotechnological Applications, Applied Microbiology and Biotechnology, vol. 99, p. 1611-1625, Jan. 11, 2015. |
| Kufer, Peter, et al. “A revival of bispecific antibodies.” Trends in biotechnology 22.5 (2004): 238-244. |
| Lee et al., “Bivalent antibody phage display mimics natural immunoglobulin,” J. Immunol. Methods, Jan. 2004, 284(1-2):119-132. |
| Lee et al., “High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold,” J. Mol. Biol., Jul. 23, 2004, 340(5):1073- 1093. |
| Lee et al. “Immunogenicity study of Globo H analogues with modification at the reducing or nonreducing end of the tumor antigen” Journal of the American Chemical Society, (2014) 136(48), 16844-16853. |
| Lehninger, Biochemistry: The Molecular Basis of Cell Structure and Function, 2nd ed., 1975, pp. 73-75, Worth Publishers, New York. |
| Leung et al., “A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction,” Technique—A Journal of Methods in Cell and Molecular Biology, Aug. 1989, 1(1):11-15. |
| Liang, Pi-Hui, et al. “Quantitative Microarray Analysis of Intact Glycolipid-CD1d Interaction and Correlation with Cell-Based Cytokine Production.” Journal of the American Chemical Society 130.37 (2008): 12348-12354. |
| Lindmark et al., “Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera,” J. Immunol. Meth., Aug. 12, 1983, 62(1):1-13. |
| Liu et al., “Eradication of large colon tumor xenografts by targeted delivery of maytansinoids,” Proc. Natl., Acad. Sci. U.S.A., Aug. 6, 1996, 93(16):8618-8623. |
| Liu, Gui, et al. “QS-21 structure/function studies: effect of acylation on adjuvant activity.” Vaccine 20.21-22 (2002): 2808-2815. |
| Livingston, Philip, “Augmenting the immunogenicity of carbohydrate tunor antigens” Seminars in Cancer Biology, Cancer Biol, 6(6):357-366, 1995. |
| Lloyd, Kenneth, “Tumor Antigens Known to be Immunogenic in Man” in Specific Immunotherapy of Cancer with Vaccines, 1993, 690, 50-58. |
| Lode et al., “Targeted therapy with a novel enediyene antibiotic calicheamicin ϑIl effectively suppresses growth and dissemination of liver metastases in a syngeneic model of murine neuroblastoma,” Cancer Res., Jul. 15, 1998, 58(14):2925-2928. |
| Lonberg et al., “Antigen-specific human antibodies from mice comprising four distinct genetic modifications,” Nature, Apr. 28, 1994, 368(6474):856-859. |
| Lonberg et al., “Human antibodies from transgenic mice,” Int. Rev. Immunol., 1995, 13(1):65-93. |
| Lou, et al., Stage-specific embryonic antigent-4 as a potential therapeutic target in glioblastoma multiforms and other cancers. Proc Natl Acad Sci USA 2014, 111(7):2482-7. |
| Lucas, A.H. et al., Carbohydrate Moieties as Vaccine Candidates: Meeting Summary, Vaccine, vol. 28(4), Jan. 2010, pp. 1121-1131. |
| Mandler et al., “Immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies: antiproliferative activity on human breast carcinoma cell lines,” J. Nat. Cancer Inst., Oct. 4, 2000, 92(19):1573-1581. |
| Mandler et al., “Modifications in synthesis strategy improve the yield and efficacy of geldanamycin-herceptin immunoconjugates,” Bioconjugate Chem., Jul.-Aug. 2002, 13(4):786-791. |
| Mandler et al., “Synthesis and evaluation of antiproliferative activity of a geldanamycin-Herceptin™ immunoconjugate,” Bioorganic & Med. Chem. Letters, May 15, 2000, 10(10):1025-1028. |
| Mao, Shenlan, et al. “Phage-display library selection of high-affinity human single-chain antibodies to tumor-associated carbohydrate antigens sialyl Lewisx and Lewisx.” Proceedings of the National Academy of Sciences 96.12 (1999): 6953-6958. |
| Mao, Weiguang, et al. “EphB2 as a therapeutic antibody drug target for the treatment of colorectal cancer.” Cancer Research 64.3 (2004): 781-788. |
| Marasco et al., “Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody,” Proc. Natl. Acad. Sci. U.S.A., Aug. 15, 1993, 90(16):7889-7893. |
| Marasco, “Intrabodies: turning the humoral immune system outside in for intracellular immunization,” Gene Therapy, Jan. 1997, 4(1):11-15. |
| Marks et al., “By-passing immunization. Human antibodies from V-gene libraries displayed on phage,” J. Mol. Biol., Dec. 5, 1991, 222(3):581-597. |
| Marks et al., “By-passing immunization: Building high affinity human antibodies by chain shuffling,” Nature Biotechnology, Jul. 1992, 10(7):779-783. |
| Martineau, R.S. et al., Immunochemical Studies on a Panosyl-Azoprotein conjugate, Immunochemistry, vol. 8, 705-718, 1971. |
| Mather et al., “Culture of testicular cells in hormone-supplemented serum-free medium,” Annals N.Y. Acad. Sci., 1982, 383:44-68. |
| Mather, “Establishment and characterization of two distinct mouse testicular epithelial cell lines,” Biol. Reprod., Aug. 1980, 23(1):243-252. |
| Matsuda, F. et al., “Structure and physical map of 64 variable segments in the 3′ 0.8-megabase region of the human immunoglobulin heavy-chain locus.” Nature Genet., 1993, 3: 88-94. |
| McCafferty et al., “Phage antibodies: Filamentous phage displaying antibody variable domains,” Nature, Dec. 6, 1990, 348:552-554. |
| Menard S et al., Generation of Monoclonal Antibodies Reacting with Normal and Cancer Cells of Human Breast, Cancer Res 43: 1295-1300, 1983. |
| Miller, Kathy, et al. “Design, construction, and in vitro analyses of multivalent antibodies.” The Journal of Immunology 170.9 (2003): 4854-4861. |
| Milstein, C & Cuello, AC, Hybrid Hydridomas and their use in immunohistochemistry, Nature 305, 537-540, Oct. 1993. |
| Morimoto et al., “Single-step purification of F(ab′)2 fragments of mouse monoclonal antibodies (immunoglobulins G1) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW,” J. Biochem. Biophys. Meth., Mar. 1992, 24(1-2):107-117. |
| Morrison et al., “Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains,” Proc. Natl. Acad. Sci. U.S.A., Nov. 1984, 81(21):6851-6855. |
| Morrison, “Immunology. Success in specification,” Nature, Apr. 28, 1994, 368(6474):812-813. |
| Munson et al., “Ligand: a versatile computerized approach for characterization of ligand-binding systems,” Anal. Biochem., Sep. 1, 1980, 107(1):220-239. |
| Neuberger et al., “Recombinant antibodies possessing novel effector functions,” Nature, Dec. 13-19, 1984, 312(5995):604-608. |
| Neuberger, “Generating high-avidity human Mabs in mice,” Nature Biotechnol., Jul. 1996, 14(7):826. |
| Nicolaou, K.C. et al., “Calicheamicin ΘI1: A rationally designed molecule with extremely potent and selective DNA cleaving properties and apoptosis inducing activity.” Angew. Chem. Intl. Ed. Engl., Feb. 1, 1994, 33(2):183-186. |
| Niculescu-Duvaz et al., “Antibody-directed enzyme prodrug therapy (Adept): A review,” Adv. Drg. Del. Rev., Jul. 7, 1997, 26(2-3):151-172. |
| Nikula, Kristen et al., Animal Models of Chronic Bronchitis and Their Relevance to Studies of Particle-Induced Disease, Inhal. Toxicol. 4(12): 123-153, 2000. |
| Office Action issued in corresponding Taiwan patent application No. 103131876, dated Dec. 26, 2016, 7 pages. |
| Orlandi et al., “Cloning immunoglobulin variable domains for expression by the polymerase chain reaction,” Proc. Natl. Acad. Sci. U.S.A., May 1989, 86(10):3833-3837. |
| Ørum et al., “Efficient method for constructing comprehensive murine Fab antibody libraries displayed on phage.” Nucleic Acids Res., Sep. 25, 1993, 21(19):4491-4498. |
| Oxenius, Annette, et al. “CpG-containing oligonucleotides are efficient adjuvants for induction of protective antiviral immune responses with T-cell peptide vaccines.” Journal of Virology 73.5 (1999): 4120-4126. |
| Papanastassiou et al., “The potential for efficacy of the modified (ICP 34.5−) herpes simplex virus HSV1716 following intratumoural injection into human malignant glioma: a proof of principle study,” Gene Therapy, Mar. 2002, 9(6):398-406. |
| Paul, William E. “Structure and Function of Immunoglobulins, Fundamental Immunology.” Chapter 9 (1993), 3rd Edition: 292-295. |
| Pearson, William, Using the FASTA Program to Search Protein and DNA Sequence Databases, Methods Mol. Biol. 243:307-331, 1994. |
| Pegram, Mark et al., Inhibitory Effects of Combinations of HER-2/neu Antibody and Chemotherapeutic Agents Used for Treatment of Human Breast Cancers, Oncogene 18:2241-2251, 1999. |
| Pegram, Mark et al., Rational Combinations of Trastuzumab With Chemotherapeutic Drugs Used in the Treatment of Breast Cancer, J. of the Nat. Cancer Inst. 96(10):739-749, 2004. |
| Plückthun, “Mono- and bivalent antibody fragments produced in Escherichia coli: Engineering, folding and antigen binding,” Immunol. Rev., Dec. 1992, 130:151-188. |
| Plückthun, Handbook of Experimental Pharmacology, vol. 113: The Pharmacology of Monoclonal Antibodies, Chapter 11: Antibodies from Escherichia coli, Rosenberg et al., eds., 1994, pp. 269-315, Springer-Verlag, Berlin. |
| Presta et al., “Humanization of an antibody directed against IgE,” J. Immunol., Sep. 1, 1993, 151(5):2623-2632. |
| Presta et al., “Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders,” Cancer Res., Oct. 15, 1997, 57(20):4593-4599. |
| Presta, Leonard G. “Antibody engineering.” Current Opinion in Biotechnology 3.4 (1992): 394-398. |
| Presta, “Antibody engineering,” Curr. Opin. Str. Biol., Aug. 1992, 2(4):593-596. |
| Proba et al., “Functional antibody single-chain fragments from the cytoplasm of Escherichia coli: influence of thioredoxin reductase (TrxB),” Gene, Jul. 4, 1995, 159(2):203-207. |
| Queen, Cary et al, A Humanized Antibody that Binds to the Interleukin 2 Receptor, Proc Natl Acad Sci., 86: 10029-10033 (1989). |
| Ragupathi, Govindaswami et al., Immunization of Mice with a Fully Synthetic Globo H Antigen Results in Antibodies against Human Cancer Cells: A Combined Chemical—Immunological Approach to the Fashioning of an Anticancer Vaccine, Angew Chem Int, 36(1-2), 125-128, Feb. 1997. |
| Ragupathi, Govindaswami, et al. “Constructing an adenocarcinoma vaccine: Immunization of mice with synthetic KH-1 nonasaccharide stimulates anti-KH-1 and anti-Ley antibodies.” International Journal of Cancer 99.2 (2002): 207-212. |
| Ramm et al., “The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. II. Isomerase-independent chaperone activity in vitro,” J. Biol. Chem., Jun. 2, 2000, 275(22):17106-17113. |
| Ravetch et al., “Fc receptors,” Annu. Rev. Immunol., 1991, 9:457-492. |
| Reyes et al., “Expression of human β-interferon cDNA under the control of a thymidine kinase promoter from herpes simplex virus,” Nature, Jun. 17, 1982, 297(5867):598-601. |
| Riechmann et al., “Reshaping human antibodies for therapy,” Nature, Mar. 24, 1988, 332(6162):323-327. |
| Rowland et al., “Drug localisation and growth inhibition studies of vindesine-monoclonal anti-CEA conjugates in a human tumour xenograft,” Cancer Immunol. Immunother., 1986, 21(3):183-187. |
| Rüde, Erwin et al., Synthesis of the N-carboxy-α-amino Acid Anhydrides of Several O-acetylated Serine Glycosides, Carbohydr. Research, 1968, 8(2), 219-232. |
| Rudikoff, Stuart, et al. “Single amino acid substitution altering antigen-binding specificity.” Proceedings of the National Academy of Sciences 79.6 (1982): 1979-1983. |
| Sastry et al., “Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library,” Proc. Natl. Acad. Sci. U.S.A., Aug. 1989, 86(15):5728-5732. |
| Schier et al., “Identification of functional and structural amino-acid residues by parsimonious mutagenesis,” Gene, Mar. 9, 1996, 169(2):147-155. |
| Schiffman, Mark, and Philip E. Castle. “The promise of global cervical-cancer prevention.” New England Journal of Medicine 353.20 (2005): 2101-2104. |
| Schwarz, Mikael, et al. “A new kind of carbohydrate array, its use for profiling antiglycan antibodies, and the discovery of a novel human cellulose-binding antibody.” Glycobiology 13.11 (2003): 749-754. |
| Search Report issued in corresponding Taiwan patent application No. 103131876, prepared Dec. 20, 2016, 1 page. |
| Sedlik, Christine et al., Effective Antitumor Therapy Based on a Novel Antibody-Drug Conjugate Targeting the Tn Carbohydrate Antigen, Oncoimmunology, Jul. 2016, vol. 5, No. 7, e1171434-1-13. |
| Shalaby et al., “Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene,” J. Exp. Med., Jan. 1, 1992, 175(1):217-225. |
| Sidhu et al., “Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions,” J. Mol. Biol., Apr. 23, 2004, 338(2):299-310. |
| Siebenlist et al., “E. coli RNA polymerase interacts homologously with two different promoters,” Cell, Jun. 1980, 20(2):269-281. |
| Sigma-Aldrich, Product Information for Hemocyanin From Megathura Crenulata, Catalog No. H7017, 1 Page, 2016. |
| Simmons et al., “Expression of full-length immunoglobulins in Escherichia coli: Rapid and efficient production of aglycosylated antibodies,” J. Immunol. Methods, May 1, 2002, 263(1-2):133-147. |
| Sims et al., “A humanized CD18 antibody can block function without cell destruction,” J. Immunol., Aug. 15, 1993, 151(4):2296-2308. |
| Sjölander, A., et al. “ISCOMs: an adjuvant with multiple functions.” J. Leukocyte Biol. 64.6 (1998): 713-723. |
| Skerra, “Bacterial expression of immunoglobulin fragments,” Curr. Opinion in Immunol., Apr. 1993, 5(2):256-262. |
| Slovin, S.F. et al., Carbohydrate Vaccines in Cancer: Immunogenicity of a Fully Synthetic Globo H Hexasaccharide Conjugate in Man, Proc Natl Acad Sci, 96:5710-5715, May 1999. |
| Sonderstrup, Grete, Development of Humanized Mice as a Model of Inflammatory Arthritis, Springer Sem. Immunopathol. 25: 35-45, 2003. |
| Speed, Margaret A., Daniel IC Wang, and Jonathan King. “Multimeric intermediates in the pathway to the aggregated inclusion body state for P22 tailspike polypeptide chains.” Protein Science 4.5 (1995): 900-908. |
| Sun, Hongfan, Kevin GJ Pollock, and James M. Brewer. “Analysis of the role of vaccine adjuvants in modulating dendritic cell activation and antigen presentation in vitro.” Vaccine 21.9-10 (2003): 849-855. |
| Suresh et al., “Bispecific monoclonal antibodies from hybrid hybridomas,” Methods in Enzymology, 1986, 121:210-228. |
| Syrigos et al., “Antibody directed enzyme prodrug therapy (ADEPT): a review of the experimental and clinical considerations,” Anticancer Research, Jan.-Feb. 1999, 19(1A):605-614. |
| Takeda et al., “Construction of chimaeric processed immunoglobulin genes containing mouse variable and human constant region sequences,” Nature, Apr. 4-10, 1985, 314(6010):452-454. |
| Thorpe, (1985) “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological and Clinical Applications, A. Pinchera et al. (Ed.s), pp. 475-506. |
| Tomlinson, I.M. et al., “The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops” J. Mol. Biol., Oct. 5, 1992, 227(3): 776-798. |
| Toyokuni, Tatsushi et al., Synthetic Vaccines: Synthesis of a Dimeric Tn Antigen-Lipopeptide Conjugate That Elicits Immune Responses Against Tn-Expressing Glycoproteins, J. Am. Chem. Soc., 1994, 116(1), 395-396. |
| Traunecker et al., “Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells,” EMBO J., Dec. 1991, 10(12):3655-3659. |
| Tutt et al., “Trispecific F(ab′)3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells,” J. Immunol., Jul. 1, 1991, 147(1):60-69. |
| Urlaub et al., “Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity.” Proc. Natl. Acad. Sci. U.S.A., Jul. 1980, 77(7):4216-4220. |
| Vajdos, Felix F., et al. “Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis.” Journal of Molecular Biology 320.2 (2002): 415-428. |
| Vaswani et al., “Humanized antibodies as potential therapeutic drugs,” Ann. Allergy, Asthma Immunol., Aug. 1998, 81(2):105-116, 119. |
| Verhoeyen et al., “Reshaping human antibodies: grafting an antilysozyme activity,” Science, Mar. 25, 1988, 239(4847):1534-1536. |
| Wakimoto, Hiroaki, et al. “Intensified antitumor immunity by a cancer vaccine that produces granulocyte-macrophage colony-stimulating factor plus interleukin 4.” Cancer Research 56.8 (1996): 1828-1833. |
| Wallner, Fredrik K., et al. “Solid-phase synthesis of serine-based glycosphingolipid analogues for preparation of glycoconjugate arrays.” Organic & Biomolecular Chemistry 3.2 (2005): 309-315. |
| Wang et al., “Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer,” Proc. Natl. Acad. Sci. U.S.A., Aug. 19, 2008, 105(33):11661-11666. |
| Ward et al., “Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli,” Nature, Oct. 12, 1989, 341(6242):544-546. |
| Waterhouse et al., “Combinatorial infection and in vivo recombination: a strategy for making large phage antibody repertoires,” Nuc. Acids Res., May 11, 1993, 21(9):2265-2266. |
| Wilen et al., “Strategies in optical resolutions,” Tetrahedron, 1977, 33(21):2725-2736. |
| Williams, S.C. and Winter, G. “Cloning and sequencing of human immunoglobulin Vλ gene segments ” Eur. J. Immunol., 1993, 23: 1456-1461. |
| Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast, 1986. |
| Winter et al., “Making antibodies by phage display technology,” Annu. Rev. Immunol., 1994, 12:433-455. |
| Wymer, Nathan et al., Enzyme-Catalyzed Synthesis of Carbohydrates, Curr. Opin. Chem. Biol., 4, 110-119, 2000. |
| Yaniv, Moshe, Enhancing Elements for Activation of Eukaryotic Promoters, Nature 297: 17-18, 1982. |
| Yansura et al., “Nucleotide sequence selection for increased expression of heterologous genes in Escherichia coli,” Methods: A Companion to Methods in Enzymol., Aug. 1992, 4(2):151-158. |
| Yelton et al., “Affinity maturation of the BR96 anti-carcinoma antibody by codon-based mutagenesis.” J. Immunol., Aug. 15, 1995, 155(4):1994-2004. |
| Zhang et al., “Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides,” Int. J. Cancer, Sep. 26, 1997,73(1):42-49. |
| Zhou, Zhifang et al., A Fully Synthetic Self-Adjuvanting Globo H-Based Vaccine Elicited Strong T Cell-Mediated Antitumor Immunity, Chem. Sci., 2015, 6, 7112-7121. |
| Zhu, Jianglong et al., From Synthesis to Biologies: Preclinical Data on a Chemistry Derived Anticancer Vaccine, J. Am. Chem. Soc. 131(26):9298-9303, 2009. |
| Eller, Chelcie et al., “Affinity of monoclonal antibodies for Globo-series glycans,” Carbohydrate Research, (2014), 397, 1-6. |
| Lou, Yi-Wei et al., “Stage-specific embryonic antigen-4 as a potential therapeutic target in glioblastoma multiforme and other cancers,” PNAS, (2014), 111(7): 2482-2487. |
| Gebauer, J Structrual Biology, vol. 128, p. 280-286, 1999. |
| Gilewski, T et al., “Immunization of of metastatic breast cancer patients with a fully synthetic globo H conjugate: A phase I trial,” PNAS, 98(6), pp. 3270-3275, Mar. 13, 2001. |
| Sasikumar et al., “Small-Molecule Immune Checkpoint INhibitors Targeting PD-1/PD-L1 and Other Emerging Checkpoint Pathways,” BioDrugs, 2018, 32:481-497. |
| BLAST alignment of GenBank AN126084.1 and SEQ ID No. 1 (downloaded Nov. 20, 2020). (Year: 2020). |
| First Examination Report dated Jul. 5, 2021 in India Patent Application No. 201717013151. |
| NCBI GenBank: AN126084.1 (submitted Jun. 8, 2016). (Year: 2016). |
| Non-Final Office Action issued in U.S. Appl. No. 14/855,260 dated Jul. 27, 2021. |
| Chang, W.W. et al., “Expression of Globo H and SSEA3 in breast cancer stem cells and the involvement of fucosyl transferases 1 and 2 in Globo H synthesis,” PNAS, Aug. 19, 2008, vol. 105 No. 33, pp. 11667-11672. |
| GenBank: CAG28308.1, May 13, 2004. |
| GenBank: CAG28309.2, Nov. 26, 2013. |
| Pravetoni, M et al. “Structurally distinct nicotine immunogens elicit antibodies with non-overlapping specificities,” Biochem Pharmacol, Feb. 15, 2012, vol. 83. No. 4, 543-550. 19 pages. |
| Sledzinska, Anna et al. “Negative immune checkpoints on T lymphocytes and their relevance to cancer immunotherapy,” Molecular Oncology, 2015, vol. 9, pp. 1936-1965 (30 pages). |
| International Search Report dated Mar. 24, 2022, in International Patent Publication No. WO 2022/072513. |
| NCT01516307—Trial of Active Immunotherapy with Globo H-KLH (OPT-822) in Metastatic Breast Cancer Subjects. Full Text Review. ClinicalTrials.gov. Jan. 2012. (https://clinicaltrials.gov/ct2/show/NCT01516307). |
| Ragupathi, G., et al., “A fully synthetic globo H carbohydrate vaccine induces a focused humoral response in prostate cancer patients: a proof of principle,” Angewandte Chemie International Edition 38.4 (1999): 569-566. Feb. 24, 1999. |
| Wang, Z.-G. et al., “Polyclonal antibodies from patients immunized with a globo H-keyhole limpet hemocyanin vaccine: Isolation, quantification, and characterization of immune responses by using totally synthetic immobilized tumor antigens,” PNAS, Mar. 14, 2000, vol. 97, No. 6, pp. 2719-2724. |
| Number | Date | Country | |
|---|---|---|---|
| 20210284719 A1 | Sep 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62368407 | Jul 2016 | US |
